U.S. patent number 9,260,503 [Application Number 14/126,215] was granted by the patent office on 2016-02-16 for multi-substituted insulins.
This patent grant is currently assigned to Novo Nordisk A/S. The grantee listed for this patent is Thomas Hoeg-Jensen, Peter Madsen, Jane Spetzler, Tina Moeller Tagmose. Invention is credited to Thomas Hoeg-Jensen, Peter Madsen, Jane Spetzler, Tina Moeller Tagmose.
United States Patent |
9,260,503 |
Hoeg-Jensen , et
al. |
February 16, 2016 |
**Please see images for:
( Certificate of Correction ) ** |
Multi-substituted insulins
Abstract
The present invention regards an insulin derivative comprising
at least 2 albumin binding moieties, wherein said albumin binding
moieties comprise fatty diacid substitutions and a method for
preparing such an insulin derivative by acylation and/or reductive
alkylation. The present invention also concern a pharmaceutical
comprising such an insulin derivative.
Inventors: |
Hoeg-Jensen; Thomas
(Klampenborg, DK), Madsen; Peter (Bagsvaerd,
DK), Spetzler; Jane (Broenshoej, DK),
Tagmose; Tina Moeller (Ballerup, DK) |
Applicant: |
Name |
City |
State |
Country |
Type |
Hoeg-Jensen; Thomas
Madsen; Peter
Spetzler; Jane
Tagmose; Tina Moeller |
Klampenborg
Bagsvaerd
Broenshoej
Ballerup |
N/A
N/A
N/A
N/A |
DK
DK
DK
DK |
|
|
Assignee: |
Novo Nordisk A/S (Bagsvaerd,
DK)
|
Family
ID: |
47356559 |
Appl.
No.: |
14/126,215 |
Filed: |
June 14, 2012 |
PCT
Filed: |
June 14, 2012 |
PCT No.: |
PCT/EP2012/061284 |
371(c)(1),(2),(4) Date: |
March 21, 2014 |
PCT
Pub. No.: |
WO2012/171994 |
PCT
Pub. Date: |
December 20, 2012 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20140228285 A1 |
Aug 14, 2014 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61499203 |
Jun 21, 2011 |
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Foreign Application Priority Data
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Jun 15, 2011 [EP] |
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11170009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
3/10 (20180101); C07K 14/62 (20130101); A61P
9/10 (20180101); A61P 9/00 (20180101); C07K
14/622 (20130101); A61P 25/02 (20180101); A61P
17/02 (20180101); C07K 1/1077 (20130101) |
Current International
Class: |
C07K
14/62 (20060101); C07K 1/107 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1254699 |
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Oct 1989 |
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JP |
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99/21578 |
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May 1999 |
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WO |
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99/22754 |
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May 1999 |
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WO |
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99/32116 |
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Jul 1999 |
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WO |
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01/00675 |
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Jan 2001 |
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WO |
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01/93837 |
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Dec 2001 |
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WO |
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2005/016312 |
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Feb 2005 |
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WO |
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2007/096431 |
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Aug 2007 |
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WO |
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2008/013938 |
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Jan 2008 |
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WO |
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2008/015099 |
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Feb 2008 |
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WO |
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2008/145721 |
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Dec 2008 |
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WO |
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2009/015456 |
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Feb 2009 |
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WO |
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2009/022005 |
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Feb 2009 |
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WO |
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2009/067636 |
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May 2009 |
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WO |
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WO 2009/115469 |
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Sep 2009 |
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WO |
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2010/029159 |
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Mar 2010 |
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WO |
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2010/080609 |
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Jul 2010 |
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WO |
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Other References
Asada et al. "Absorption Characteristics of Chemically
Modified-Insulin Derivatives with Various Fatty Acids in the Small
and Large Intestine," Journal of Pharmaceutical Sciences, Jun.
1995, vol. 84, pp. 682-687. cited by examiner .
Asada H et al., Stability of acyl derivatives of insulin in the
small intestine: Relative importance of insulin association
characteristics in aqueous solution, Pharmaceutical Research, 1994,
vol. 11 (8), 1115-1120. cited by applicant .
D. G. Lindsay et al., Acetoacetylation of insulin, The Biochemical
Journal, 1969, vol. 115(3), 587-595. cited by applicant .
D. G. Lindsay et al., The Acetylation of Insulin, Biochemical
Journal, 1971, vol. 121, 737-745. cited by applicant .
Ehrat M. et al., Synthesis and Spectroscopic Characterization of
Insulin Derivatives Containing One or Two Poly (ethylene oxide)
Chains at Specific Positions, Biopolymers, 1983, vol. 22, 569-573.
cited by applicant .
Friesen Heinz-Jurgen et al., Preparation and Application of Nalpha
-B1, N epsilon-B29-bis(tert.-butyloxycarbonyl) insulin,
Hoppe-Seyler's Z. Physiological Chemistry, 1978, vol. 359, 103-111.
cited by applicant .
Hashimoto M et al., Synthesis of palmitoyl derivatives of insulin
and their biological activities, Pharmaceutical Research, 1989,
vol. 6(2), 171-176. cited by applicant .
Hashizume M et al., Improvement of large intestinal absorption of
insulin by chemical modification with palmitic acid in rats, The
Journal of pharmacy and pharmacology, 1992, vol. 44(7), 555-559.
cited by applicant .
Lindsay D.G. et al., Carbamyl- and methylthiocarbamylinsulins,
Biochimica et Biophysica Acta (BBA)--Protein Structure, 1972, vol.
263 (3), 658-665. cited by applicant .
Shozo Muranishi et al., Trials of lipid modification of peptide
hormones for intestinal delivery, Journal of Controlled Release,
1992, vol. 19, Issues 1-3, 179-188. cited by applicant .
Takashi Uchio et al., Site-specific insulin conjugates with
enhanced stability and extended action profile, Advanced Drug
Delivery Reviews, 1999, vol. 35, Issues 2-3, 289-306. cited by
applicant .
Ulla Ribel et al., Equivalent In Vivo Biological Activity of
Insulin Analogues and Human Insulin Despite Different In Vitro
Potencies, Diabetes, 1990, vol. 39, 1033-1039. cited by applicant
.
Yogish C Kudva et al., Ultra-long-acting insulins for a
lifestyle-related pandemic, The Lancet, 2011, vol. 377(9769),
880-881. cited by applicant.
|
Primary Examiner: Bradley; Christina
Attorney, Agent or Firm: Hu; Jianjie
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. .sctn.371 National Stage
application of International Application PCT/EP2012/061284
(WO2012/171994), filed Jun. 14, 2012, which claimed priority of
European Patent Application 11170009.2, filed Jun. 15, 2011; this
application claims priority under 35 U.S.C. .sctn.119 of U.S.
Provisional Application 61/499,203; filed Jun. 21, 2011.
Claims
The invention claimed is:
1. A soluble insulin derivative or pharmaceutically acceptable salt
thereof selected from the group consisting of
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethy-
l-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethy-
l-benzyl A14E B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl
B1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethy-
l-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-be-
nzyl
B1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl A14E B25H desB30 human insulin,
B1N-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-benzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B16H B25H desB30human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl A14E B25H
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
desB30human insulin, and
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B25H desB27 desB30 human insulin, wherein OEG is
*--NH--(CH.sub.2).sub.2O--(CH.sub.2).sub.2O--CH.sub.2CO--*.
2. A soluble insulin derivative or a pharmaceutically acceptable
salt thereof of general formula XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1,
wherein Ins represents an insulin comprising a B29 lysine or B29
arginine residue and/or a A22 lysine residue, X is a fatty diacid
substitution, X.sup.1 is a fatty diacid substitution, Z is a linker
between X and Ins, Z.sup.1 is a linker between Ins and X.sup.1, n
is zero or 1, and m is zero or 1, and wherein at least one of Z and
Z.sup.1 comprises OEG-OEG-aminomethyl-benzyl, wherein OEG is
*--NH--(CH.sub.2).sub.2O--(CH.sub.2).sub.2O--CH.sub.2CO--*.
3. The soluble insulin derivative or a pharmaceutically acceptable
salt thereof according to claim 2, wherein Ins represents an
insulin comprising a B29 lysine or B29 arginine residue and a A22
lysine residue, and X is located at said A22 lysine residue.
4. The soluble insulin derivative or a pharmaceutically acceptable
salt thereof according to claim 3, wherein said fatty diacid
substitutions each comprise 14-20 carbon atoms.
5. The soluble insulin derivative or a pharmaceutically acceptable
salt thereof according to claim 4, wherein X.sup.1 is located at a
position selected from the group consisting of B29 lysine,
N-terminus of the A chain, and N-terminus of the B chain.
6. The soluble insulin derivative or a pharmaceutically acceptable
salt thereof according to claim 4, wherein X.sup.1 is located at a
position selected from the group consisting of N-terminus of the A
chain, and N-terminus of the B chain.
7. The soluble insulin derivative or a pharmaceutically acceptable
salt thereof according to claim 4, wherein X.sup.1 is located at a
position selected from the group consisting of B29lysine and
N-terminus of the B chain.
8. The soluble insulin derivative or pharmaceutically acceptable
salt thereof according to claim 3, wherein said fatty acid
substitutions are each selected from a group of protracting
moieties selected from Chem. 3 and Chem. 4, wherein Chem 3 is
HOOC--(CH.sub.2).sub.x--CO--*, and Chem 4 is
HOOC--C.sub.6H4-O--(CH.sub.2).sub.y--CO--*, wherein x is an integer
from 10 to 20 and y is an integer from 6 to 14.
9. The soluble insulin derivative or pharmaceutically acceptable
salt thereof according to claim 8, wherein x is 14, 16 or 18 and y
is 8, 10 or 12.
10. The soluble insulin derivative or pharmaceutically acceptable
salt thereof according to claim 5, wherein said fatty acid
substitutions are each selected from a group of protracting
moieties selected from Chem. 3 and Chem. 4, wherein Chem 3 is
HOOC--(CH.sub.2)x-CO--*, and Chem 4 is
HOOC--C.sub.6H4-O--(CH.sub.2)y-CO--*, wherein x is 14, 16 or 18 and
y is 8, 10 or 12.
11. The soluble insulin derivative or pharmaceutically acceptable
salt thereof according to claim 10, wherein Z and Z.sup.1 comprise
one or more linker elements selected from the group consisting of
selected from the group consisting of: alpha-L-Glu, alpha-D-Glu,
gamma-L-Glu, gamma-D-Glu, alpha-L-Asp, alpha-D-Asp, beta-L-Asp,
beta-D-Asp, CPH, IDA and OEG, wherein CPH is
*--CH.sub.2PhCH.sub.2NH--*, IDA is
*--N((CH.sub.2).sub.nCOOH)(CH.sub.2).sub.mCO--*, wherein n is 1 or
2 and m is 1 or 2, and OEG is
*--NH--(CH.sub.2).sub.2O--(CH.sub.2).sub.2O--CH.sub.2CO--*, wherein
at least one of Z and Z.sup.1 comprises
OEG-OEG-aminomethyl-benzyl.
12. A pharmaceutical composition comprising a soluble insulin
derivative or a pharmaceutically acceptable salt thereof of claim 2
and a pharmaceutically suitable excipient.
13. A pharmaceutical composition comprising a soluble insulin
derivative or a pharmaceutically acceptable salt thereof of claim 1
and a pharmaceutically suitable excipient.
14. A method of treating hyperglycemia, type 2 diabetes, impaired
glucose tolerance, or type 1 diabetes in a subject in need of said
treatment, said method comprising administering to a said subject a
therapeutically effective amount of a pharmaceutical composition of
claim 12.
15. A method of treating hyperglycemia, type 2 diabetes, impaired
glucose tolerance, or type 1 diabetes in a subject in need of said
treatment, said method comprising administering to a said subject a
therapeutically effective amount of a pharmaceutical composition of
claim 13.
Description
TECHNICAL FIELD
The present invention relates to novel insulin derivatives which
are useful in the treatment of diabetes and related aspects.
BACKGROUND
In mammals, insulin lowers blood glucose and is used for treatment
of diabetes type 1 and type 2, with the goal of adjusting blood
glucose towards healthy levels. In healthy persons, blood glucose
levels are regulated close to 5 mM during the fasting state,
whereas values up towards 10 mM can occur for a few hours after a
meal. Blood glucose levels are influenced by many factors such as
timing and character of meals and insulin administrations,
exercise, infections and more. Blood glucose can fluctuate widely
and unpredictably in diabetes patients and can fluctuate in one
patient in the range 1-30 mM.
Diabetes patients benefit from a constant supply of basal insulin
drug, because native insulin is quickly cleared from the body. In
order to limit the number of injections required for maintaining
basal insulin levels, insulin has been engineered with various
prolongation principles, such as crystallizations or chemical
derivatisations.
Reversible binding to circulating proteins such as serum albumin
can also be a factor prolonging the in vivo activity of drugs.
Albumin binding as a protraction principle has been exploited for
insulin and other peptides by conjugation of the drug with fatty
acids, fatty diacids or related compounds, optionally incorporated
via various linkers.
Low affinity insulin analogs have been shown to give rise to an
equivalent total effect on glucose utilization as high affinity
analogs in euglycemic glucose-clamp studies in pigs, suggesting
that the insulin biological activity can be similar for both low-
and high-affinity analogues. However the low affinity analogs
exerted their effect over a longer time period when compared to the
high affinity analogues (See e.g. Ribel, U., et. al. Equivalent in
vivo biological activity of insulin analogues and human insulin
despite different in vitro potencies. Diabetes 39, 1033-1039
(1990), abstract attached).
WO1999/032116 and WO1999/021578 regard fatty acid-acylated
insulins, WO1999/022754, WO1999/032116, WO1999/021578, JP1254699
administering a fatty acid di- and triacylated insulin or insulin
analogue by inhalation, U.S. Pat. No. 3,868,356 concerns acylation
of insulins with dicarboxylic acid functional derivatives, e.g.
anhydrides.
There is still a need for basal insulin drugs with a prolonged in
vivo activity.
SUMMARY
The present invention regards an insulin derivative comprising two
or more substitutions, each comprising a fatty diacid substitution,
the insulin derivative optionally comprises a linker between the
insulin and the fatty acid substitution.
The present invention also regards a method for preparing such an
insulin derivative or a pharmaceutical salt thereof by acylation
and/or reductive alkylation of an insulin.
The present invention regards an insulin derivative for the
treatment of diabetes or related aspects and may thus also be used
as a medicament.
DESCRIPTION
The present invention regards an insulin, substituted with at least
two albumin binding moieties, each comprising a protracting moiety,
which more specifically is a fatty diacid substitution. Optionally
the albumin binding moiety further comprises various linkers. The
derivatisation of an insulin according to this invention is
achieved by acylation and/or reductive alkylation, obtaining an
increased albumin affinity and in vivo circulation times of the
resulting insulin derivative relative to native human insulin.
The present invention also provides a method for reductive
alkylation of the B1 residue (e.g. the N-terminal of the B-chain of
the insulin).
The present invention also provides a method for reductive
alkylation of the A1 residue (e.g. the N-terminal of the A-chain of
the insulin).
The object of this invention is to overcome or ameliorate at least
one of the disadvantages of the prior art, or to provide a useful
alternative.
The invention may also solve further problems that will be apparent
from the disclosure of the exemplary embodiments.
It has been found, that substitution of insulins or insulin
analogues with at least two fatty diacids enables insulin binding
to human serum albumin and prolongs the in vivo insulin supply,
retaining IR-binding properties. This is favourable, since
substitutions according to this invention are also possible on
insulin analogues that have only very few (e.g. 2-6) mutations and
thus this invention provides the option of prolonging the insulin
in vivo supply of insulin and maintaining an insulin backbone that
is very similar to the native human insulin.
In one aspect of the present invention the affinity of insulin
derivatives substituted with at least two fatty diacids to albumin
is increased when compared to single substituted insulin
derivatives.
In one aspect of the present invention the prolongation effect is
increased when compared to single substituted insulin
derivatives.
The insulin derivatives of this invention are long-acting, and in
one aspect they show increased tendency to oligomerisation in the
subcutaneous depot, enabling slow diffusion to the circulation.
In one aspect the affinity of an insulin derivative according to
the present invention to albumin is elevated with increasing fatty
diacid chain length.
In one aspect the affinity of an insulin derivative according to
the present invention to albumin is elevated with increasing fatty
diacid chain up to 22 carbon atoms.
In one aspect the insulin derivative is maintained overall
hydrophilic with increasing fatty diacid chain up to 22 carbon
atoms.
In one aspect the prolongation effect of at least two albumin
binding moieties according to this invention is elevated with
increasing fatty diacid chain length.
In one aspect the prolongation effect of at least two albumin
binding moieties according to this invention is elevated with
increasing fatty diacid chain up to 22 carbon atoms.
In one embodiment an insulin derivative according to the present
invention comprises at fatty diacid substitution consisting of up
to 22 carbon atoms. In one embodiment an insulin derivative
according to the present invention comprises at fatty diacid
substitution consisting of 10-22 carbon atoms. In one embodiment an
insulin derivative according to the present invention comprises at
fatty diacid substitution consisting of 10-20 carbon atoms. In one
embodiment an insulin derivative according to the present invention
comprises at fatty diacid substitution consisting of 10-18 carbon
atoms. In one embodiment an insulin derivative according to the
present invention comprises at fatty diacid substitution consisting
of 10-16 carbon atoms. In one embodiment an insulin derivative
according to the present invention comprises at fatty diacid
substitution consisting of 10-14 carbon atoms. In one embodiment an
insulin derivative according to the present invention comprises at
fatty diacid substitution consisting of 12-20 carbon atoms. In one
embodiment an insulin derivative according to the present invention
comprises at fatty diacid substitution consisting of 12-18 carbon
atoms. In one embodiment an insulin derivative according to the
present invention comprises at fatty diacid substitution consisting
of 12-16 carbon atoms. In one embodiment an insulin derivative
according to the present invention comprises at fatty diacid
substitution consisting of 12-14 carbon atoms. In one embodiment an
insulin derivative according to the present invention comprises at
fatty diacid substitution consisting of 14-20 carbon atoms. In one
embodiment an insulin derivative according to the present invention
comprises at fatty diacid substitution consisting of 14-18 carbon
atoms. In one embodiment an insulin derivative according to the
present invention comprises at fatty diacid substitution consisting
of 14-16 carbon atoms. In one embodiment an insulin derivative
according to the present invention comprises at fatty diacid
substitution consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
or 20 carbon atoms. In one embodiment an insulin derivative
according to the present invention comprises at fatty diacid
substitution consisting of 14, 15, 16, 17, 18, 19 or 20 carbon
atoms. In one embodiment an insulin derivative according to the
present invention comprises at fatty diacid substitution consisting
of 16, 17, 18, 19 or 20 carbon atoms.
In one embodiment of the present invention an insulin derivative
comprises at least 2 albumin binding moieties, wherein said albumin
binding moieties comprise fatty diacid substitutions and wherein
one carboxy group from each of said fatty diacid substitutions are
attached, optionally via a linker, to an insulin.
In one embodiment of the present invention an insulin derivative
comprises at least 3 albumin binding moieties, wherein said albumin
binding moieties comprise fatty diacid substitutions and wherein
one carboxy group from each of said fatty diacid substitutions are
attached, optionally via a linker, to an insulin.
In one embodiment of the present invention an insulin derivative
comprises 2 or 3 albumin binding moieties, wherein said albumin
binding moieties comprise fatty diacid substitutions and wherein
one carboxy group from each of said fatty diacid substitutions are
attached, optionally via a linker, to an insulin.
In one embodiment of the present invention an insulin derivative
comprises 2 albumin binding moieties, wherein said albumin binding
moieties comprise fatty diacid substitutions and wherein one
carboxy group from each of said fatty diacid substitutions are
attached, optionally via a linker, to an insulin.
In one embodiment of the present invention an insulin derivative
comprises 2 albumin binding moieties, wherein said albumin binding
moieties comprise fatty diacid substitutions and wherein one
carboxy group from each of said fatty diacid substitutions are
attached, optionally via a linker, to amino acid residues in an
insulin.
In one embodiment of the present invention an insulin derivative
comprises 2 albumin binding moieties, wherein said albumin binding
moieties comprise fatty diacid substitutions and wherein one
carboxy group from each of said fatty diacid substitutions are
attached, optionally via a linker, to amino acid residues in an
insulin located in any of the following positions: A22, B29 or the
N-terminal amino acid residue of the A chain of an insulin and/or
the N-terminal amino acid residue of the B chain of an insulin.
In one embodiment of the present invention an insulin derivative
comprises 3 albumin binding moieties, wherein said albumin binding
moieties comprise fatty diacid substitutions and wherein one
carboxy group from each of said fatty diacid substitutions are
attached, optionally via a linker, to an insulin.
In one embodiment of the present invention an insulin derivative
comprises 3 albumin binding moieties, wherein said albumin binding
moieties comprise fatty diacid substitutions and wherein one
carboxy group from each of said fatty diacid substitutions are
attached, optionally via a linker, to amino acid residues in an
insulin.
In one embodiment of the present invention an insulin derivative
comprises 3 albumin binding moieties, wherein said albumin binding
moieties comprise fatty diacid substitutions and wherein one
carboxy group from each of said fatty diacid substitutions are
attached, optionally via a linker, to amino acid residues in an
insulin located in any of the following positions: A22, B29, the
N-terminal amino acid residue of the A chain of an insulin and/or
the N-terminal amino acid residue of the B chain of an insulin.
In one embodiment of the present invention an insulin derivative is
represented by the general formula Chem 1:
##STR00001##
in which Ins represents an insulin comprising a B29 lysine or B29
arginine residue and/or a A22 lysine residue, X, X.sup.1 and
X.sup.2 is a fatty diacid substitution and X.sup.2 is optional, Z,
Z.sup.1 and Z.sup.2 is a linker between Ins and X, X.sup.1 and
X.sup.2, respectively and n, m and p is zero or one.
In one embodiment of the present invention an insulin derivative is
represented by the general formula Chem 1, in which Ins represents
an insulin comprising a B29 lysine or B29 arginine residue and/or a
A22 lysine residue, X, X.sup.1 and X.sup.2 is a fatty diacid
substitution and respectively located in a position selected from
the group consisting of: B29 lysine, A22 lysine, N-terminal of the
A chain, N-terminal of the B-chain, Z, Z.sup.1 and Z.sup.2 is a
linker between Ins and X, X.sup.1 and X.sup.2, respectively and n,
m and p is zero or one.
In one embodiment of the present invention an insulin derivative is
represented by the general formula Chem. 2:
XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, Chem 2
in which Ins represents an insulin comprising a B29 lysine or B29
arginine residue and/or a A22 lysine residue, X and X.sup.1 is a
fatty diacid substitution, Z and Z.sup.1 is a linker between Ins
and X and X.sup.1 respectively, and n and m is zero or one.
In one embodiment of the present invention an insulin derivative is
represented by the general formula Chem. 2, in which Ins represents
an insulin comprising a B29 lysine or B29 arginine residue and/or a
A22 lysine residue, X and X.sup.1 is a fatty diacid substitution
and respectively located in a position selected from the group
consisting of: B29 lysine, A22 lysine, N-terminal of the A chain,
N-terminal of the B-chain, Z and Z.sup.1 is a linker between Ins
and X and X.sup.1 respectively, and n and m is zero or one.
When used herein "Z, Z.sup.1 and Z.sup.2 is a linker between Ins
and X, X.sup.1 and X.sup.2, respectively" or "Z and Z.sup.1 is a
linker between Ins and X and X.sup.1 respectively" means that Z,
Z.sup.1 or Z.sup.2 is a linker between the amino acid in the
insulin (Ins) and the respective protraction moiety X, X.sup.1 or
X.sup.2. More specifically this means, that Z is a linker between
the insulin (Ins) and the protracting moiety X, Z.sup.1 is a linker
between the insulin (Ins) and the protracting moiety X.sup.1 and
Z.sup.2 is a linker between the insulin (Ins) and the protracting
moiety X.sup.2.
The letters n, m and p of Chem. 1 and Chem. 2 independently
represent the number of linkers (Z, Z.sup.1 and Z.sup.2,
respectively) represented in the insulin derivative and n, m and p
is zero or one. More specifically this means that when n is zero,
no linker is present between the insulin (Ins) and the protraction
moiety (X) in Chem. 1 or Chem. 2, when m is zero, no linker is
present between the insulin (Ins) and the protraction moiety
(X.sup.1) in Chem. 1 or Chem. 2 an when p is zero, no linker is
present between the insulin (Ins) and the protraction moiety
(X.sup.2) in Chem. 1, when n is one, one linker is present between
the insulin (Ins) and the protraction moiety (X) in Chem. 1 or
Chem. 2, when m is one, one linker is present between the insulin
(Ins) and the protraction moiety (X.sup.1) in Chem. 1 or Chem. 2 an
when p is one, one linker is present between the insulin (Ins) and
the protraction moiety (X.sup.2) in Chem. 1.
The albumin binding moiety (e.g. Z.sub.nX in formula Chem. 1 and
Chem. 2), the protracting moiety (e.g. X in formula Chem. 1 and
Chem. 2) or the linker (e.g. Z in formula Chem. 1 and Chem. 2) may
be covalently attached to a lysine residue or the N-terminal of the
A and/or B chain of the insulin by acylation and/or reductive
alkylation.
In one embodiment an insulin analogue comprises less than 10 amino
acid modifications (substitutions, deletions, additions (including
insertions) and any combination thereof) relative to human insulin,
alternatively less than 9, 8, 7, 6, 5, 4, 3, 2 or 1 modifications
relative to human insulin.
Modifications in the insulin molecule are denoted stating the chain
(A or B), the position, and the one or three letter code for the
amino acid residue substituting the native amino acid residue.
In one embodiment an insulin derivative according to this invention
comprises at least 2 albumin binding moieties.
In one embodiment an albumin binding moiety (e.g. Z.sub.nX Chem. 1)
of an insulin derivative according to the present invention
comprises a protracting moiety, which may also be designated fatty
diacid substitution (e.g. X in Chem. 1).
In one embodiment each albumin binding moiety of an insulin
derivative according to the present invention comprises a
protracting moiety and optionally a linker (e.g. Z.sub.n in Chem.
1, wherein n is one).
In one embodiment, an activated ester of the albumin binding
moiety, preferably comprising a protracting moiety and a linker, is
covalently linked to an amino group of a lysine residue, preferably
the epsilon amino group thereof, under formation of an amide bond
(this process being referred to as acylation).
In one embodiment, an activated ester of the albumin binding moiety
(e.g. Z.sub.nX, Chem. 1), preferably comprising a protracting
moiety (e.g. X, Chem. 1) and a linker (e.g. Z.sub.n wherein n is
one, Chem. 1), is covalently linked to an amino group of a lysine
residue and/or an amino acid residue in the N-terminal of the A- or
B-chain, preferably the epsilon amino group thereof, under
formation of an amide bond (this process being referred to as
acylation).
In one embodiment, an aldehyde derivative of the albumin binding
moiety, preferably comprising a protracting moiety and a linker, is
covalently linked by reductive alkylation to the alpha-amino group
in the N-terminal of the A-chain or the alpha-amino group in the
N-terminal of the B-chain or aldehyde derivatives are reductively
alkylated at an N-terminal amino acid residue at A-chain and/or the
B-chain.
In one embodiment, an aldehyde derivative of the albumin binding
moiety, preferably comprising a protracting moiety and a linker, is
covalently linked by reductive alkylation to a lysine residue,
preferably the epsilon-amino group thereof.
In one embodiment an insulin according to the present invention
comprises an arginine residue in position B29 and is substituted
with an albumin binding moiety at the A22 lysine and not at the B29
position.
In one embodiment an insulin derivative according to this invention
is desired to comprise one albumin binding moiety at the A22
position, one albumin binding moiety at another amino acid
positions in the insulin and no albumin binding moiety at the B29
position, the insulin subject to substitution comprises an arginine
residue in the B29 position.
In one embodiment an insulin derivative according to this invention
is desired to comprise one albumin binding moiety at the A22
position, and one albumin binding moiety at an other amino acid
positions of the insulin, such as the B29 position of the insulin,
the insulin subject to substitution may comprise a lysine residue
in the B29 position.
In one embodiment an insulin according to the present invention is
substituted with an albumin binding moiety at the A22 lysine and
the B29 lysine.
In one embodiment, each albumin binding moiety (e.g. Z.sub.nX,
Chem. 1) comprises a protracting moiety (e.g. X) independently
selected from Chem. 3, and Chem. 4: HOOC--(CH.sub.2).sub.x--CO--*
Chem. 3 HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem.
4
in which x is an integer in the range of 10-20, y is an integer in
the range of 6-14.
In one embodiment X of Chem. 1 or Chem. 2 is selected from Chem. 3,
and Chem. 4: HOOC--(CH.sub.2).sub.x--CO--* Chem. 3
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem. 4
in which x is an integer in the range of 10-20, y is an integer in
the range of 6-14.
In one embodiment X.sup.1 of Chem. 1 or Chem. 2 is selected from
Chem. 3, and Chem. 4: HOOC--(CH.sub.2).sub.x--CO--* Chem. 3
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem. 4
in which x is an integer in the range of 10-20, y is an integer in
the range of 6-14.
In one embodiment X.sup.2 of Chem. 2 is selected from Chem. 3, and
Chem. 4: HOOC--(CH.sub.2).sub.x--CO--* Chem. 3
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem. 4
in which s is an integer in the range of 10-20, t is an integer in
the range of 6-14.
In one embodiment X, X.sup.1 and X.sup.2 of Chem. 1 or Chem. 2 are
independently selected from Chem. 3, and Chem. 4:
HOOC--(CH.sub.2).sub.x--CO--* Chem. 3
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem. 4
in which x is an integer in the range of 10-20, y is an integer in
the range of 6-14.
In one embodiment, *--(CH.sub.2).sub.x--* of Chem. 3 refers to
straight or branched, preferably straight, alkylene in which x is
an integer in the range of 10-20.
In another embodiment, *--(CH.sub.2).sub.y--* of Chem. 4 refers to
straight or branched, preferably straight, alkylene in which y is
an integer in the range of 6-14.
In one embodiment, *--(CH.sub.2).sub.x--* of Chem. 3 refers to
straight or branched, preferably straight, alkylene in which x is
an integer in the range of 12-20.
In another embodiment, *--(CH.sub.2).sub.y--* Chem. 4 refers to
straight or branched, preferably straight, alkylene in which y is
an integer in the range of 6-12.
In one embodiment, *--(CH.sub.2).sub.x--* of Chem. 3 refers to
straight or branched, preferably straight, alkylene in which x is
an integer in the range of 14-20.
In another embodiment, *--(CH.sub.2).sub.y--* of Chem. 4 refers to
straight or branched, preferably straight, alkylene in which y is
an integer in the range of 6-10.
In one embodiment, *--(CH.sub.2).sub.x--* of Chem. 3 refers to
straight or branched, preferably straight, alkylene in which x is
an integer in the range of 14-16.
In another embodiment, *--(CH.sub.2).sub.y--* Chem. 4 refers to
straight or branched, preferably straight, alkylene in which y is
an integer in the range of 6-8.
In one embodiment, *--(CH.sub.2).sub.x--* of Chem. 3 refers to
straight or branched, preferably straight, alkylene in which x is
an integer in the range of 12-18.
In another embodiment, *--(CH.sub.2).sub.y--* of Chem. 4 refers to
straight or branched, preferably straight, alkylene in which y is
an integer in the range of 8-10.
In one embodiment, *--(CH.sub.2).sub.x--* of Chem. 3 refers to
straight or branched, preferably straight, alkylene in which x is
an integer in the range of 14-18.
In another embodiment, *--(CH.sub.2).sub.y--* Chem. 4 refers to
straight or branched, preferably straight, alkylene in which y is
an integer in the range of 8-12.
In one embodiment, *--(CH.sub.2).sub.x--* of Chem. 3 refers to
straight or branched, preferably straight, alkylene in which x is
an integer in the range of 10-16.
In another embodiment, *--(CH.sub.2).sub.y--* Chem. 4 refers to
straight or branched, preferably straight, alkylene in which y is
an integer in the range of 8-14.
In one embodiment, *--(CH.sub.2).sub.x--* of Chem. 3 refers to
straight or branched, preferably straight, alkylene in which x is
an integer and selected from the group consisting of 12, 14, 16, 18
and 20.
In another embodiment, *--(CH.sub.2).sub.y--* Chem. 4 refers to
straight or branched, preferably straight, alkylene in which y is
an integer and selected from the group consisting of 6, 8, 10, 12
and 14.
In one embodiment, *--(CH.sub.2).sub.x--* of Chem. 3 refers to
straight or branched, preferably straight, alkylene in which x is
an integer and selected from the group consisting of 14, 16, 18 and
20.
In another embodiment, *--(CH.sub.2).sub.y--* Chem. 4 refers to
straight or branched, preferably straight, alkylene in which y is
an integer and selected from the group consisting of 8, 10 and
12.
In one embodiment one of the acid groups of the fatty diacid forms
an amide bond with the epsilon amino group of a lysine residue in
said insulin, preferably via a linker.
The term "insulin subject to substitution" when used herein, means
the insulin that is treated by the method provided herein and thus
an insulin which is substituted with an albumin binding moiety,
resulting in an insulin derivative according to this invention.
The term "fatty diacid" refers to fatty acids with an additional
carboxylic acid group in the omega position. Thus, fatty diacids
are dicarboxylic acids.
The nomenclature is as is usual in the art, for example in the
above formulas *--COOH as well as HOOC--* refers to carboxy;
*--C.sub.6H.sub.4--* to phenylene; *--CO--*, as well as *--OC--*,
to carbonyl (O.dbd.C<**).
In particular embodiments, the aromatics, such as the phenoxy, and
the phenylene radicals, may be, independently, ortho, meta, or
para.
In one embodiment an insulin derivative according to this invention
comprises at least 2 albumin binding moieties, each albumin binding
moiety comprises a protracting moiety selected from Chem. 3, or
4.
In one embodiment an insulin derivative according to this invention
comprises at least 2 albumin binding moieties, each albumin binding
moiety comprises a protracting moiety selected from Chem. 3, or 4
and the albumin binding moiety optionally further comprises a
linker, wherein each linker comprises one ore more linker element
of formula Chem. 5, Chem. 6, Chem. 7, Chem. 8, Chem. 9, Chem. 10,
and/or Chem. 11.
In one embodiment an insulin derivative according to this invention
comprises at least 2 albumin binding moieties, each albumin binding
moiety comprises a protracting moiety selected from Chem. 3, or 4
and the albumin binding moiety further comprises a linker (which
may be designated Z).
In one embodiment a linker comprises one or more linker elements of
formula Chem. 5, Chem. 6, Chem. 7, Chem. 8, Chem. 9, Chem. 10,
and/or Chem. 11.
In one embodiment a linker element according to this invention may
be designated e.
In one embodiment a linker (Z) comprises two linker elements, which
may be designated e.sub.1-e.sub.2, indicating the arrangement
relative to each other, e.g. linker element e.sub.1 is attached to
linker element e.sub.2.
In one embodiment a linker (Z) comprises three linker elements,
which may be designated e.sub.1-e.sub.2-e.sub.3, indicating the
arrangement relative to each other, e.g. linker element e.sub.1 is
attached to linker element e.sub.2 and linker element e.sub.2 is
attached to linker element e.sub.1 and linker element e.sub.3.
In one embodiment a linker (Z) comprises four linker elements,
which may be designated e.sub.1-e.sub.2-e.sub.3-e.sub.4, indicating
the arrangement relative to each other, e.g. linker element e.sub.1
is attached to linker element e.sub.2 and linker element e.sub.2 is
attached to linker element e.sub.1 and linker element e.sub.3 and
linker element e.sub.3 is attached to linker element e.sub.4.
In one embodiment a linker (Z) comprises four linker elements,
which may be designated e.sub.1-e.sub.2-e.sub.3-e.sub.4-e.sub.5,
indicating the arrangement relative to each other, e.g. linker
element e.sub.1 is attached to linker element e.sub.2 and linker
element e.sub.2 is attached to linker element e.sub.1 and linker
element e.sub.3, linker element e.sub.3 is attached to linker
element e.sub.4 and e.sub.4 is attached to linker element
e.sub.5.
In one embodiment a linker (Z) comprises four linker elements,
which may be designated
e.sub.1-e.sub.2-e.sub.3-e.sub.4-e.sub.5-e.sub.6, indicating the
arrangement relative to each other, e.g. linker element e.sub.1 is
attached to linker element e.sub.2 and linker element e.sub.2 is
attached to linker element e.sub.1 and linker element e.sub.3,
linker element e.sub.3 is attached to linker element e.sub.4,
e.sub.4 is attached to linker element e.sub.5 and e.sub.5 is
attached to linker element e.sub.6.
In one embodiment a linker element of a linker according to the
present invention, designated with the highest number (e.g. e.sub.4
in the linker e.sub.1-e.sub.2-e.sub.3-e.sub.4 or e.sub.3 in the
linker e.sub.1-e.sub.2-e.sub.3) is attached to a protracting moiety
(i.e. a fatty diacid).
A linker of the derivative of the invention may comprise the
following first linker element:
##STR00002##
wherein k is an integer in the range of 1-5, and n is an integer in
the range of 1-5.
In a particular embodiment, when k=1 and n=1, this linker element
may be designated OEG, or a di-radical of 8-amino-3,6-dioxaoctanic
acid, and/or it may be represented by the following formula:
*--NH--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--CH.sub.2--CO--*.
Chem. 5a
In one embodiment, each linker of the derivative of the invention
further comprise, independently or in combination with one or more
other linker element, a second linker element, preferably a Glu
di-radical, such as Chem. 6 and/or Chem. 7:
##STR00003##
wherein the Glu di-radical may be included p times, where p is an
integer in the range of 1-3.
Chem. 6 may also be referred to as gamma-Glu, or briefly g-Glu, due
to the fact that it is the gamma carboxy group of the amino acid
glutamic acid which is here used for connection to another linker
element, or to the epsilon-amino group of lysine. The other linker
element may, for example, be another Glu residue, or an OEG
molecule. The amino group of Glu in turn forms an amide bond with
the carboxy group of the protracting moiety, or with the carboxy
group of, e.g., an OEG molecule, if present, or with the
gamma-carboxy group of, e.g., another Glu, if present.
Chem. 7 may also be referred to as alpha-Glu, or briefly aGlu, or
simply Glu, due to the fact that it is the alpha carboxy group of
the amino acid glutamic acid which is here used for connection to
another linker element, or to the epsilon-amino group of lysine or
to the N-terminal of the A-chain or B-chain.
The above structures of Chem. 6 and Chem. 7 cover the L-form, as
well as the D-form of Glu. The L-form may be designated gamma-L-Glu
or gLGlu, whereas the D-form may be designated gamma-D-Glu or
gDGlu. In particular embodiments, Chem. 6 and/or Chem. 7 is/are,
independently, a) in the L-form, or b) in the D-form.
In one embodiment, each linker of the derivative of the invention
further comprise, independently or in combination with one or more
other linker element, a second linker element, preferably a Asp
di-radical, such as Chem. 8 and/or Chem. 9:
##STR00004##
wherein the Asp di-radical may be included p times, where p is an
integer in the range of 1-3.
Chem. 8 may also be referred to as beta-Asp, or briefly bAsp, due
to the fact that it is the gamma carboxy group of the amino acid
aspartic acid which is here used for connection to another linker
element, or to the epsilon-amino group of lysine or to the
N-terminal of the A-chain or B-chain. The other linker element may,
for example, be another Asp residue, or an OEG molecule. The amino
group of Asp in turn forms an amide bond with the carboxy group of
the protracting moiety, or with the carboxy group of, e.g., an OEG
molecule, if present, or with the beta-carboxy group of, e.g.,
another Asp, if present.
Chem. 9 may also be referred to as alpha-Asp, or briefly aAsp, or
simply Asp, due to the fact that it is the alpha carboxy group of
the amino acid aspartic acid which is here used for connection to
another linker element, or to the epsilon-amino group of
lysine.
The above structures of Chem. 8 and Chem. 9 cover the L-form, as
well as the D-form of Asp. The L-form may be designated beta-L-Asp
or bLAsp, whereas the D-form may be designated beta-D-Asp or bDAsp.
In particular embodiments, Chem. 8 and/or Chem. 9 is/are,
independently, a) in the L-form, or b) in the D-form.
In one embodiment, each linker of the derivative of the invention
further comprise, independently or in combination with other linker
elements, the following third linker element:
*--N((CH.sub.2).sub.nCOOH)(CH.sub.2).sub.mCO--*, n=1-2 Chem. 10
in which n and m is an integer in the range of 1-2. This linker
element may be designated IDA.
In one embodiment, each linker of the derivative of the invention,
when the albumin binding moiety is attached by reductive alkylation
further comprise, independently or in combination with other linker
elements, the following linker element: *--CH.sub.2PhCH.sub.2NH--*
Chem. 11
This linker element may be designated CPH.
In still further particular embodiments the linker has a) from 5 to
41 C-atoms; and/or b) from 4 to 28 hetero atoms. Particular and
non-limiting examples of hetero atoms are N-, and O-atoms. H-atoms
are not hetero atoms.
Alternatively, the linker moiety, if present, has from 5 to 30
C-atoms, preferably from 5 to 25 C-atoms, more preferably from 5 to
20 C-atoms, or most preferably from 5 to 17 C-atoms. In additional
preferred embodiments, the linker moiety, if present, has from 4 to
20 hetero atoms, preferably from 4 to 18 hetero atoms, more
preferably from 4 to 14 hetero atoms, or most preferably from 4 to
12 hetero atoms.
Alternatively, the linker comprises at least one OEG molecule,
and/or at least one glutamic acid residue, or rather the
corresponding radicals.
In one embodiment, each linker consists of one time Chem. 6 and two
times Chem. 5, interconnected via amide bonds and in the sequence
indicated, the linker being connected at its free amino end to the
free carbonyl group of the protracting moiety, and at its free
carbonyl end to a B29 lyseine residue, a A22K lysine residue or the
N-terminal of the A and/or B chain of an insulin.
In one embodiment one or more albumin binding moieties are attached
to an insulin by acylation.
In one embodiment one or more albumin binding moieties are attached
to an insulin by reductive alkylation.
One embodiment is a method for substituting an insulin or insulin
derivative according to this invention with an albumin binding
moiety by reductive alkylation.
In one embodiment an insulin is substituted according to this
invention in two steps, in a first step one or more albumin binding
moieties are attached to an insulin by acylation and in a second
step one or more albumin binding moieties are attached by reductive
alkylation to the insulin derivative achieved in the first
step.
In one embodiment an insulin is substituted according to this
invention in two steps, in a first step one or more albumin binding
moieties are attached to an insulin by reductive alkylation and in
a second step one or more albumin binding moieties are attached by
acylation to the insulin derivative achieved in the first step.
In one embodiment an amine in the N-terminal of the A and/or B
chain of the inulin reacts with an aldehyde function in the albumin
binding moiety.
In one embodiment an insulin or insulin derivative according to
this invention is substituted with an albumin binding moiety by
reductive alkylation.
In one embodiment an insulin or insulin derivative according to
this invention is substituted with one or more albumin binding
moieties, by reductive alkylation using a reducing agent.
In one embodiment an insulin or insulin derivative according to
this invention is substituted with one or more albumin binding
moieties, by reductive alkylation using NaCNBH.sub.3 as reducing
agent.
One embodiment is a method for substituting an insulin or insulin
derivative according to this invention with an albumin binding
moiety by reductive alkylation, wherein NaCNBH.sub.3 is used as
reducing agent.
One embodiment is a method for substituting an insulin or insulin
derivative according to this invention with an albumin binding
moiety at said insulin's N-terminal amino acid residue in said
insulin's A and/or B chain.
One embodiment of the present invention is a method for
substituting an insulin or insulin derivative according to this
invention with an albumin binding moiety at said insulin's
N-terminal amino acid residue in said insulin's A and/or B chain,
wherein NaCNBH.sub.3 is used as reducing agent.
In one embodiment, the at least two side chains of the present
invention are similar.
In one embodiment, the at least two albumin binding moieties of the
present invention (i.e. the entire side chains) are similar.
In one embodiment, the protracting moieties of each albumin binding
moiety are similar.
The term "similar" as used herein, referring to the at least two
side chains or albumin binding moieties of the present invention,
means that the combination of protracting moieties and linkers are
the same (e.g. Z=Z.sup.1, n=m and X=X.sup.1, Chem. 1).
The term "similar" as used herein, referring to the protracting
moieties of the at least two side chains of the present invention,
means that the combination of protracting moieties are the same in
the side chains of the insulin derivative (e.g. X=X.sup.1).
The term "similar" as used herein, referring to the protracting
moieties of the at least two albumin binding moieties of the
present invention, means that the combination of protracting
moieties are the same in the albumin binding moieties of the
insulin derivative (e.g. X=X.sup.1).
In one embodiment, the combination of linker elements of each side
chain or albumin binding moiety are similar (e.g. for one insulin
derivative according to this invention, if
e.sub.1-e.sub.2=gDGlu-aLAsp combination for Z, e.sub.1-e.sub.2 for
Z.sub.1 is also a gDGlu-aLAsp combination).
In one embodiment, the combination of linker elements in each side
chain or albumin binding moiety are not similar (e.g. for one
insulin derivative according to this invention, if e.sub.1-e.sub.2
is a gDGlu-aLAsp combination for Z, e.sub.1-e.sub.2 for Z.sub.1 is
another combination of linker elements than a gDGlu-aLAsp
combination, e.g. gDGlu-OEG).
In one embodiment, the combination of linker elements in the
linkers (if present) of the at least two side chains of the present
invention are not similar (e.g. for one insulin derivative
according to this invention, if e.sub.1-e.sub.2 is a gDGlu-aLAsp
combination for Z, e.sub.1-e.sub.2 for Z.sub.1 is another
combination of linker elements than a gDGlu-aLAsp combination, e.g.
gDGlu-OEG) and the protracting moiety are not similar (e.g. X is
not the same fatty diacid as X.sub.1).
In one embodiment, the insulin derivative according to this
invention is in the form of a pharmaceutically acceptable salt.
In one embodiment the salt may be a basic salt, an acid salt, or it
may be neither nor (i.e. a neutral salt). Basic salts produce
hydroxide ions and acid salts produce hydronium ions in water.
The salts of the insulin derivatives of the invention may be formed
with added cations or anions that react with anionic or cationic
groups, respectively. These groups may be situated in the peptide
moiety, and/or in the side chain of the derivatives of the
invention.
Non-limiting examples of anionic groups of the derivatives of the
invention include free carboxylic groups in the side chain, if any,
as well as in the peptide moiety. The peptide moiety often includes
a free carboxylic acid group at the C-terminus, and it may also
include free carboxylic groups at internal acid amino acid residues
such as Asp and Glu.
Non-limiting examples of cationic groups in the peptide moiety
include the free amino group at the N-terminus, if present, as well
as any free amino group of internal basic amino acid residues such
as His, Arg, and Lys.
In one embodiment, an insulin derivative according to the invention
is used as a pharmaceutical.
In one embodiment, an insulin derivative according to the invention
is used as a medicament.
In one embodiment, an insulin derivative according to the invention
is used as a medicament for delaying or preventing disease
progression in type 2 diabetes.
In one embodiment of the invention, the insulin derivative is for
use as a medicament for the treatment or prevention of
hyperglycemia including stress induced hyperglycemia, type 2
diabetes, impaired glucose tolerance, type 1 diabetes, and burns,
operation wounds and other diseases or injuries where an anabolic
effect is needed in the treatment, myocardial infarction, stroke,
coronary heart disease and other cardiovascular disorders is
provided.
In one embodiment, the invention is related to a method for the
treatment or prevention of hyperglycemia including stress induced
hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1
diabetes, and burns, operation wounds and other diseases or
injuries where an anabolic effect is needed in the treatment,
myocardial infarction, coronary heart disease and other
cardiovascular disorders, stroke, the method comprising
administering to a patient in need of such treatment an effective
amount for such treatment of an insulin derivative according to the
invention.
The term "diabetes" or "diabetes mellitus" includes type 1
diabetes, type 2 diabetes, gestational diabetes (during pregnancy)
and other states that cause hyperglycaemia. The term is used for a
metabolic disorder in which the pancreas produces insufficient
amounts of insulin, or in which the cells of the body fail to
respond appropriately to insulin thus preventing cells from
absorbing glucose. As a result, glucose builds up in the blood.
Type 1 diabetes, also called insulin-dependent diabetes mellitus
(IDDM) and juvenile-onset diabetes, is caused by B-cell
destruction, usually leading to absolute insulin deficiency.
Type 2 diabetes, also known as non-insulin-dependent diabetes
mellitus (NIDDM) and adult-onset diabetes, is associated with
predominant insulin resistance and thus relative insulin deficiency
and/or a predominantly insulin secretory defect with insulin
resistance.
In one embodiment, an insulin derivative according to the invention
is used for the preparation of a medicament for the treatment or
prevention of hyperglycemia including stress induced hyperglycemia,
type 2 diabetes, impaired glucose tolerance, type 1 diabetes,
burns, operation wounds, other diseases or injuries where an
anabolic effect is needed in the treatment, myocardial infarction,
stroke, coronary heart disease, other cardiovascular disorders,
treatment of critically ill diabetic and non-diabetic patients and
polyneuropathy.
The term "human insulin" as used herein means the human insulin
hormone whose structure and properties are well-known. Human
insulin has two polypeptide chains, named the A-chain and the
B-chain. The A-chain is a 21 amino acid peptide and the B-chain is
a 30 amino acid peptide, the two chains being connected by
disulphide bridges: a first bridge between the cysteine in position
7 of the A-chain and the cysteine in position 7 of the B-chain, and
a second bridge between the cysteine in position 20 of the A-chain
and the cysteine in position 19 of the B-chain. A third bridge is
present between the cysteines in position 6 and 11 of the
A-chain.
In the human body, the hormone is synthesized as a single-chain
precursor proinsulin (preproinsulin) consisting of a prepeptide of
24 amino acids followed by proinsulin containing 86 amino acids in
the configuration: prepeptide-B-Arg Arg-C-Lys Arg-A, in which C is
a connecting peptide of 31 amino acids. Arg-Arg and Lys-Arg are
cleavage sites for cleavage of the connecting peptide from the A
and B chains.
"An insulin" according to the invention is herein to be understood
as human insulin or an insulin from another species such as porcine
or bovine insulin.
"A soluble insulin" according to the invention is herein to be
understood as an insulin which is soluble in an aqueous solutions,
including but not limited to water.
In one embodiment an insulin according to the present invention is
soluble in water. In one embodiment an insulin according to the
present invention is soluble aqueous solutions. In one embodiment
an insulin according to the present invention is soluble in aqueous
solutions with a pH ranging from pH 6 to 9. In one embodiment an
insulin according to the present invention is soluble in aqueous
solutions with a pH ranging from pH 7 to 8. In one embodiment an
insulin according to the present invention is soluble in aqueous
solutions with a pH ranging from pH 7.2 to 7.8. In one embodiment
an insulin according to the present invention is soluble aqueous
solutions with a pH ranging from pH 7.2 to 7.6. In one embodiment
an insulin according to the present invention is soluble in aqueous
solutions with a pH ranging from pH 7.4 to 7.6. In one embodiment
an insulin according to the present invention is soluble in aqueous
solutions with a pH ranging from pH 7.4 to 7.8. In one embodiment
an insulin according to the present invention is soluble in pH
basic aqueous solutions. In one embodiment an insulin according to
the present invention is soluble in pH neutral aqueous solutions.
In one embodiment an insulin according to the present invention is
soluble in aqueous solutions being neutral or 1-2 pH units below
neutral. In one embodiment an insulin according to the present
invention is soluble in aqueous solutions being neutral or 1 pH
units below neutral.
In one embodiment an insulin according to this invention has a
solubility of between 0.5 mM and 8 mM. In one embodiment an insulin
according to this invention has a solubility of between 0.6 mM and
7.2 mM. In one embodiment an insulin according to this invention
has a solubility of at least 0.6 mM. In one embodiment an insulin
according to this invention has a solubility of at least 0.8 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 1.0 mM. In one embodiment an insulin
according to this invention has a solubility of at least 1.2 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 1.4 mM. In one embodiment an insulin
according to this invention has a solubility of at least 1.8 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 2.0 mM. In one embodiment an insulin
according to this invention has a solubility of at least 2.2 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 2.4 mM. In one embodiment an insulin
according to this invention has a solubility of at least 2.6 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 2.8 mM. In one embodiment an insulin
according to this invention has a solubility of at least 3.0 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 3.2 mM. In one embodiment an insulin
according to this invention has a solubility of at least 3.4 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 3.6 mM. In one embodiment an insulin
according to this invention has a solubility of at least 3.8 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 4.0 mM. In one embodiment an insulin
according to this invention has a solubility of at least 4.2 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 4.4 mM. In one embodiment an insulin
according to this invention has a solubility of at least 4.6 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 4.8 mM. In one embodiment an insulin
according to this invention has a solubility of at least 5.0 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 5.2 mM. In one embodiment an insulin
according to this invention has a solubility of at least 5.4 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 5.6 mM. In one embodiment an insulin
according to this invention has a solubility of at least 5.8 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 6.0 mM. In one embodiment an insulin
according to this invention has a solubility of at least 6.2 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 6.4 mM. In one embodiment an insulin
according to this invention has a solubility of at least 6.6 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 6.8 mM. In one embodiment an insulin
according to this invention has a solubility of at least 7.0 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 7.2 mM. In one embodiment an insulin
according to this invention has a solubility of at least 7.4 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 7.6 mM. In one embodiment an insulin
according to this invention has a solubility of at least 7.8 mM. In
one embodiment an insulin according to this invention has a
solubility of at least 8.0 mM.
The term "insulin peptide" as used herein means a peptide which is
either human insulin or an analogue or a derivative thereof with
insulin activity.
The term "insulin derivative" as used herein means a chemically
modified insulin, wherein the modification(s) are in the form of
attachment of amides, carbohydrates, alkyl groups, acyl groups,
esters, PEGylations, and the like. Examples of derivatives of human
insulin according to the invention are
A22N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A22K desB30
human insulin,
A22N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A22K
desB30 human insulin,
A22N.sup..epsilon.-tetradecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-tetradecandioyl-.gamma.-L-glutamyl A22K desB30
human insulin, A22N.sup..epsilon.-octadecandioyl-.gamma.-D-glutamyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-D-glutamyl A22K desB30
human insulin, A22N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A14E A22K B25H
desB30 human insulin,
A22N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
A22K B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A14E B25H
desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl desB30 human
insulin, A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl A14E B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl--
benzyl
B1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminom-
ethyl-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup.a-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-be-
nzyl A14E B25H desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl
B29N.sup..epsilon.octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-N-carboxymethyl-beta-alanyl
B29N.sup..epsilon.-octadecandioyl-N-carboxymethyl-beta-alanyl A14E
B25H desB30 human insulin,
A1N.sup..alpha.octadecandioyl-N-2-carboxyethyl-glycyl
B29N.sup..epsilon.-octadecandioyl-N-2-carboxyethyl-glycyl A14E B25H
desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-N-carboxymethyl-beta-alanyl
A22N.sup..epsilon.--N-octadecandioyl-N-carboxymethyl-beta-alanyl
A22K B29R desB30 human insulin,
A22N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B16H B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E B25H
B29R desB30 human,
A22N.sup..epsilon.-eicosandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-eicosandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
A22K B25H desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B16H B25H desB30 human insulin,
B1N.sup..alpha.-(octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl--
benzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B25H desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B25H desB27 desB30 human insulin,
A22N.sup..epsilon.-4-carboxyphenoxy-decanoyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-4-carboxyphenoxy-decanoyl-.gamma.-L-glutamyl-OEG-OEG
A14E A22K B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-gamma-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-gamma-L-glutamyl-OEG-OEG A14E
B16H B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-gamma-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-gamma-L-glutamyl-OEG-OEG A14E
B16H desB27 desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-amino-
methyl-benzyl A14E B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-amino-
methyl-benzyl A14E B25H desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
desB27 desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-OEG-OEG-4-amino-
methyl-benzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-OEG-OEG
A14E desB27 desB30 human insulin.
The term "insulin analogue" as used herein means a modified human
insulin wherein one or more amino acid residues of the insulin have
been substituted by other amino acid residues and/or wherein one or
more amino acid residues have been deleted from the insulin and/or
wherein one or more amino acid residues have been added and/or
inserted to the insulin.
In one embodiment an insulin analogue comprises less than 10 amino
acid modifications (substitutions, deletions, additions (including
insertions) and any combination thereof) relative to human insulin,
alternatively less than 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification
relative to human insulin.
Modifications in the insulin molecule are denoted stating the chain
(A or B), the position, and the one or three letter code for the
amino acid residue substituting the native amino acid residue.
By "connecting peptide" or "C-peptide" is meant a connection moiety
"C" of the B-C-A polypeptide sequence of a single chain
proinsulin-molecule. In the human insulin chain, the C-peptide
connects position 30 of the B chain and position 1 of the A chain
and is 35 amino acid residue long. The connecting peptide includes
two terminal dibasic amino acid sequence, e.g., Arg-Arg and Lys-Arg
which serve as cleavage sites for cleavage off of the connecting
peptide from the A and B chains to form the two-chain insulin
molecule.
By "desB30" or "B(1-29)" is meant a natural insulin B chain or an
analogue thereof lacking the B30 amino acid and "A(1-21)" means the
natural insulin A chain. Thus, e.g., A14E A22K desB30 human insulin
is an analogue of human insulin where the amino acid in position 14
in the A chain is substituted with glutamic acid, the amino acid in
position 22 in the A chain is substituted with lysine, and the
amino acid in position 30 in the B chain is deleted.
Herein terms like "A1", "A2" and "A3" etc. indicates the amino acid
in position 1, 2 and 3 etc., respectively, in the A chain of
insulin (counted from the N-terminal end). Similarly, terms like
B1, B2 and B3 etc. indicates the amino acid in position 1, 2 and 3
etc., respectively, in the B chain of insulin (counted from the
N-terminal end). Using the one letter codes for amino acids, terms
like A21A, A21G and A21Q designates that the amino acid in the A21
position is A, G and Q, respectively. Using the three letter codes
for amino acids, the corresponding expressions are A21Ala, A21Gly
and A21Gln, respectively.
Herein, the term "amino acid residue" is an amino acid from which,
formally, a hydroxy group has been removed from a carboxy group
and/or from which, formally, a hydrogen atom has been removed from
an amino group.
Amino acids exist in the stereoisomeric form of either D (dextro)
or L (levo). The D and L refer to the absolute confirmation of
optically active compounds. With the exception of glycine, all
other amino acids are mirror images that can not be superimposed.
Most of the amino acids found in nature are of the L-type. Hence,
eukaryotic proteins are always composed of L-amino acids although
D-amino acids are found in bacterial cell walls and in some peptide
antibiotics. At least 300 amino acids have been described in nature
but only twenty of these are typically found as components in human
peptides and proteins. Twenty standards amino acids are used by
cells in peptide biosynthesis, and these are specified by the
general genetic code. The twenty standard amino acids are Alanine
(Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile), Phenylalanine
(Phe), Tryptophan (Trp), Methionine (Met), Proline (Pro), Apartic
acid (Asp), Gltamic acid (Glu), Glycine (Gly), Serine (Ser),
Threonine (Thr), Cysteine (Cys), Tyrosine (Tyr), Apsagine (Asn),
Glutamine (Gln), Lysine (Lys), Arginine (Arg) and Histidine
(His).
Examples of insulin analogues are such wherein Tyr (Y) in position
14 of the A chain is substituted with Glu (E) and/or Lys (K) at
position B29 is substituted with Pro (P), Arg (R). Furthermore, Asn
(N) at position B3 may be substituted with Lys (K). Also one or
more amino acids may be added to the C-terminal of the A-chain
and/or B-chain such as, e.g., Lys (K). The amino acid in position
B1 may be substituted with Glu (E). The amino acid in position B16
may be substituted with His (H). Further examples of insulin
analogues are the deletion analogues, e.g., analogues where the B30
amino acid in human insulin has been deleted (desB30 human
insulin), desB28-B30 human insulin and desB27 human insulin.
Insulin analogues wherein the A-chain and/or the B-chain have an
N-terminal extension and insulin analogues wherein the A-chain
and/or the B-chain have a C-terminal extension such as with two
arginine residues added to the C-terminal of the B-chain are also
examples of insulin analogues. Further examples are insulin
analogues comprising combinations of the mentioned mutations.
Insulin analogues wherein the amino acid in position B25 is His(H)
and which optionally further comprises one or more additional
mutations are further examples of insulin analogues. Insulin
analogues of human insulin wherein the amino acid residue in
position A22 is Lys (K) and/or wherein the insulin analogue is
further extended in the C-terminal with two Arg (R) residues are
also examples of insulin analogues.
Further examples of insulin analogues include: desB30 human
insulin, A22K desB30 human insulin, A14E A22K desB30 human insulin,
A14E A22K B25H B29R desB30 human insulin, A14E A22K B25H desB30
human insulin A14E B25H desB27 desB30 human insulin, A14E B25H
desB30 human insulin, A14E B16H desB30 human insulin, A14E B16H
B25H desB30 human insulin B28D human insulin, A22K B29R desB30
human insulin, B3K B28E human insulin, B28D desB30 human insulin,
A22K B29P desB30 human insulin, B28K B29P human insulin, B28K B29P
desB30 human insulin, B3K B28E desB30 human insulin, A14E desB27
desB30 human insulin and A14E B16H desB27 desB30 human insulin
Insulin Receptor Binding Assay (HIRspa):
The affinity of the insuloin derivatives of this invention for the
human insulin receptor is determined by a SPA assay (Scintillation
Proximity Assay) microtiterplate antibody capture assay. SPA-PVT
antibody-binding beads, anti-mouse reagent (Amersham Biosciences,
Cat No. PRNQ0017) are mixed with 25 mL of binding buffer (100 mM
HEPES pH 7.8; 100 mM sodium chloride, 10 mM MgSO.sub.4, 0.025%
Tween-20). Reagent mix for a single Packard Optiplate (Packard No.
6005190) is composed of 2.4 .mu.l of a 1:5000 diluted purified
recombinant human insulin receptor (either with or without exon
11), an amount of a stock solution of A14Tyr[.sup.125I]-human
insulin corresponding to 5000 cpm per 100 .mu.l of reagent mix, 12
.mu.l of a 1:1000 dilution of F12 antibody, 3 mL of SPA-beads and
binding buffer to a total of 12 mL. A total of 100 .mu.l reagent
mix is then added to each well in the Packard Optiplate and a
dilution series of the insulin derivative is made in the Optiplate
from appropriate samples. The samples are then incubated for 16
hours while gently shaken. The phases are the then separated by
centrifugation for 1 min and the plates counted in a Topcounter.
The binding data were fitted using the nonlinear regression
algorithm in the GraphPad Prism 2.01 (Graph Pad Software, San
Diego, Calif.) and affinities are expressed relative (in percentage
(%)) to the affinity of human insulin.
A related assay is also used wherein the binding buffer also
contains 1.5% HSA in order to mimic physiological conditions
Pharmacokinetics Assay, Intravenous Rat PK:
Anaesthetized rats are dosed intravenously (i.v.) with insulin
derivatives at various doses and plasma concentrations of the
employed compounds are measured using immunoassays or mass
spectrometry at specified intervals for 4 hours or more post-dose.
Pharmacokinetic parameters are subsequently calculated using
WinNonLin Professional (Pharsight Inc., Mountain View, Calif.,
USA).
Non-fasted male Wistar rats (Taconic) weighing approximately 200
gram are used.
Body weight is measured and rats are subsequently anaesthetized
with Hypnorm/Dormicum (each compound is separately diluted 1:1 in
sterile water and then mixed; prepared freshly on the experimental
day). Anesthesia is initiated by 2 mL/kg Hypnorm/Doricum mixture sc
followed by two maintenance doses of 1 mL/kg sc at 30 min intervals
and two maintenance doses of 1 mL/kg sc with 45 min intervals. If
required in order to keep the rats lightly anaesthetized throughout
a further dose(s) 1-2 mL/kg sc is supplied. Weighing and initial
anaesthesia is performed in the rat holding room in order to avoid
stressing the animals by moving them from one room to another.
Albumin Binding Assay, Retention Time (RT)
Measurements of drug-protein binding by using immobilized human
serum albumin chromatography-mass spectrometry.
Albumin binding, measured by LC-MS as retention time (Rt) on
immobilized HSA-column.
The solvent was used in the following order:
A: 50 mM Ammonium Acetate pH 7.4 (3,854 g/1 L) freshly prepared
B: 100% 2-propanol
TABLE-US-00001 Time A solvent B solvent flow (min) (%) (%) (mL/min)
0 100 0 0.7 5 65 35 0.7 15 60 40 0.7 16 100 0 0.7 20 100 0 0.7
The HPLC 1100 system (CTC PAL autosampler) was aligned as
follows:
HPLC-Column: Chiral HSA 50.times.3.0 mm 5 .mu.m (Chromtech cat no:
HSA 50.3 06-F)
UV detector: 280 nm
Column Temperature: 45.degree. C.
Compound injection: 10 .mu.L, 10 .mu.M
Split 1:4 (MS:LC)
The LC/MSD Trap XCT was aligned as follows:
Ion Source Type: ESI
Polarity: Positive
Dry Temp: 325.degree. C.
Nebulizer: 40.00 psi
Dry Gas: 8.00 L/min
Production of Insulin
The production of polypeptides, e.g., insulins, is well known in
the art. The insulin or insulin analogue used as part of the
insulin derivative may for instance be produced by classical
peptide synthesis, e.g., solid phase peptide synthesis using t-Boc
or Fmoc chemistry or other well established techniques, see, e.g.,
Greene and Wuts, "Protective Groups in Organic Synthesis", John
Wiley & Sons, 1999. The insulin or insulin analogue may also be
produced by a method which comprises culturing a host cell
containing a DNA sequence encoding the analogue and capable of
expressing the insulin or insulin analogue in a suitable nutrient
medium under conditions permitting the expression of the insulin or
insulin analogue. Several recombinant methods may be used in the
production of human insulin and human insulin analogues. Examples
of methods which may be used in the production of insulin in
microorganisms such as, e.g., Escherichia coli and Saccharomyces
cerevisiae are, e.g., disclosed in WO2008/034881.
Typically, the insulin or insulin analogue is produced by
expressing a DNA sequence encoding the insulin or insulin analogue
in question or a precursor thereof in a suitable host cell by
well-known technique as disclosed in e.g. EP1246845 or
WO2008/034881.
The insulin or insulin analogue may be expressed with an N-terminal
extension as disclosed in EP 1,246,845. After secretion to the
culture medium and recovery, the insulin precursor will be
subjected to various in vitro procedures to remove the possible
N-terminal extension sequence and connecting peptide to give the
insulin or insulin analogue. Such methods include enzymatic
conversion by means of trypsin or an Achromobacter lyticus protease
in the presence of an L-threonine ester followed by conversion of
the threonine ester of the insulin or insulin analogue into insulin
or insulin analogue by basic or acid hydrolysis as described in
U.S. Pat. No. 4,343,898 or U.S. Pat. No. 4,916,212
Examples of N-terminal extensions of the type suitable in the
present invention are disclosed in U.S. Pat. No. 5,395,922 and
EP0765395.
For insulin analogues comprising non-natural amino acid residues,
the recombinant cell should be modified such that the non-natural
amino acids are incorporated into the analogue, for instance by use
of tRNA mutants. Hence, briefly, the insulin or insulin analogue
according to the invention are prepared analogously to the
preparation of known insulin analogues.
Protein Purification
The insulin or insulin analogue used as part of the insulin
derivative of the invention are recovered from the cell culture
medium. The insulin or insulin analogue of the present invention
may be purified by a variety of procedures known in the art
including, but not limited to, chromatography (e.g., ion exchange,
affinity, hydrophobic, chromatofocusing, and size exclusion),
electrophoretic procedures (e.g., preparative isoelectric focusing
(IEF), differential solubility (e.g., ammonium sulfate
precipitation), or extraction (see, e.g., Protein Purification,
J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York,
1989). Preferably, they may be purified by affinity chromatography
on an anti-insulin or anti-insulin analogue antibody column.
Additional purification may be achieved by conventional chemical
purification means, such as high performance liquid chromatography.
Other methods of purification, including barium citrate
precipitation, are known in the art, and may be applied to the
purification of the novel insulin or insulin analogue described
herein (see, for example, Scopes, R., Protein Purification,
Springer-Verlag, N.Y., 1982).
Pharmaceutical Formulations
Pharmaceutical compositions containing an insulin derivative
according to the present invention may be administered to a patient
in need of such treatment at several sites, for example, at topical
sites, for example, skin and mucosal sites, at sites which bypass
absorption, for example, administration in an artery, in a vein, in
the heart, and at sites which involve absorption, for example,
administration in the skin, under the skin, in a muscle or in the
abdomen.
Administration of pharmaceutical compositions according to the
invention may be through several routes of administration, for
example, lingual, sublingual, buccal, in the mouth, oral, in the
stomach and intestine, nasal, pulmonary, for example, through the
bronchioles and alveoli or a combination thereof, epidermal,
dermal, transdermal, vaginal, rectal, ocular, for examples through
the conjunctiva, uretal, and parenteral to patients in need of such
a treatment.
Compositions of the current invention may be administered in
several dosage forms, for example, as solutions, suspensions,
emulsions, microemulsions, multiple emulsion, foams, salves,
pastes, plasters, ointments, tablets, coated tablets, rinses,
capsules, for example, hard gelatine capsules and soft gelatine
capsules, suppositories, rectal capsules, drops, gels, sprays,
powder, aerosols, inhalants, eye drops, ophthalmic ointments,
ophthalmic rinses, vaginal pessaries, vaginal rings, vaginal
ointments, injection solution, in situ transforming solutions, for
example in situ gelling, in situ setting, in situ precipitating, in
situ crystallization, infusion solution, and implants.
For parenteral administration, an insulin derivative of this
invention is formulated analogously with the formulation of known
insulins. Furthermore, for parenterally administration, an insulin
derivative of this invention is administered analogously with the
administration of known insulins and the physicians are familiar
with this procedure.
Parenteral administration can be performed by means of a syringe,
optionally a pen-like syringe. Alternatively, parenteral
administration can be performed by means of an infusion pump.
Injectable compositions containing an insulin derivative of this
invention can be prepared using the conventional techniques of the
pharmaceutical industry which involve dissolving and mixing the
ingredients as appropriate to give the desired end product. Thus,
according to one procedure, an insulin derivative of this invention
is dissolved in an amount of water which is somewhat less than the
final volume of the composition to be prepared. An isotonic agent,
a preservative and a buffer is added as required and the pH value
of the solution is adjusted, if necessary, using an acid, for
example, hydrochloric acid, or a base, for example, aqueous sodium
hydroxide, as needed. Finally, the volume of the solution is
adjusted with water to give the desired concentration of the
ingredients.
Formulations intended for oral use may be prepared according to any
known method, and such formulations may contain one or more agents
selected from the group consisting of sweetening agents, flavouring
agents, colouring agents, and preserving agents in order to provide
pharmaceutically elegant and palatable preparations. Tablets may
contain the active ingredient in a mixture with non-toxic
pharmaceutically-acceptable excipients which are suitable for the
manufacture of tablets. The tablets may be uncoated or they may be
coated by known techniques to delay disintegration or release of
the therapeutically active polypeptide.
The orally administrable formulations of the present invention may
be prepared and administered according to methods well known in
pharmaceutical chemistry, see Remington's Pharmaceutical Sciences,
17.sup.th ed. (A. Osol ed., 1985).
The insulin derivative preparations of this invention are used
similarly to the use of the known insulin preparations.
The Following is a Non-Limiting List of Aspect Further Comprised
within the Scope of the Invention:
1) An insulin derivative or a pharmaceutically acceptable salt
thereof comprising at least 2 albumin binding moieties, wherein
each albumin binding moiety comprises a fatty diacid substitution
and wherein one carboxy group from each of said fatty diacid
substitutions is attached, optionally via a linker, to an insulin.
2) An insulin derivative or a pharmaceutically acceptable salt
thereof comprising at least 3 albumin binding moieties, wherein
each albumin binding moiety comprises a fatty diacid substitution
and wherein one carboxy group from each of said fatty diacid
substitutions is attached, optionally via a linker, to an insulin.
3) An insulin derivative or a pharmaceutically acceptable salt
thereof comprising 2 or 3 albumin binding moieties, wherein each
albumin binding moiety comprises a fatty diacid substitution and
wherein one carboxy group from each of said substitutions is
attached, optionally via a linker, to an insulin. 4) An insulin
derivative or a pharmaceutically acceptable salt thereof comprising
2 albumin binding moieties, wherein each albumin binding moiety
comprises a fatty diacid substitution and wherein one carboxy group
from each of said fatty diacid substitutions is attached,
optionally via a linker, to an insulin. 5) An insulin derivative or
a pharmaceutically acceptable salt comprising 3 albumin binding
moieties, wherein each albumin binding moiety comprises a fatty
diacid substitution and wherein one carboxy group from each of said
fatty diacid substitutions is attached, optionally via a linker, to
an insulin. 6) An insulin derivative or a pharmaceutically
acceptable salt thereof, according to any one of the previous
aspects, comprising at least 3 albumin binding moieties, wherein
each albumin binding moiety comprises a fatty diacid substitution
and wherein one carboxy group from each of said fatty diacid
substitutions is attached, optionally via a linker, to an insulin.
7) An insulin derivative or a pharmaceutically acceptable salt
thereof, according to any one of the previous aspects comprising 2
or 3 albumin binding moieties, wherein each albumin binding moiety
comprises a fatty diacid substitution and wherein one carboxy group
from each of said substitutions is attached, optionally via a
linker, to an insulin. 8) An insulin derivative or a
pharmaceutically acceptable salt thereof, according to any one of
the previous aspects, comprising 2 albumin binding moieties,
wherein each albumin binding moiety comprises a fatty diacid
substitution and wherein one carboxy group from each of said fatty
diacid substitutions is attached, optionally via a linker, to an
insulin. 9) An insulin derivative or a pharmaceutically acceptable
salt thereof, according to any one of the previous aspects,
comprising 3 albumin binding moieties, wherein each albumin binding
moiety comprises a fatty diacid substitution and wherein one
carboxy group from each of said fatty diacid substitutions is
attached, optionally via a linker, to an insulin. 10) An insulin
derivative or a pharmaceutically acceptable salt thereof of the
general formula
##STR00005## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and X.sup.2 is
optional c. Z, Z.sup.1 and Z.sup.2 is a linker between Ins and X,
X.sup.1 and X.sup.2, respectively and d. n, m and p is zero or 1.
11) An insulin derivative or a pharmaceutically acceptable salt
thereof according to any of the preceding aspects of the general
formula
##STR00006## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and X.sup.2 is
optional c. Z, Z.sup.1 and Z.sup.2 is a linker between Ins and X,
X.sup.1 and X.sup.2, respectively and d. n, m and p is zero or 1.
12) An insulin derivative or a pharmaceutically acceptable salt
thereof according to any of the preceding aspects of the general
formula
##STR00007## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue, b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and respectively
located in a position selected from the group consisting of: B29
lysine, A22 lysine, N-terminal of the A chain, N-terminal of the
B-chain. c. Z, Z.sup.1 and Z.sup.2 is a linker between Ins and X,
X.sup.1 and X.sup.2, respectively and d. n, m and p is zero or 1.
13) An insulin derivative or a pharmaceutically acceptable salt
thereof according to any of the preceding aspects of the general
formula
##STR00008## wherein a. Ins represents an insulin comprising a B29
arginine residue and a A22 lysine residue, b. X, X.sup.1 and
X.sup.2 is a fatty diacid substitution, and X is located at said
A22 lysine residue and X.sup.1 is located the N-terminal of the A
chain and X.sup.2 is not present, c. Z, Z.sup.1 and Z.sup.2 is a
linker between Ins and X, X.sup.1 and X.sup.2, respectively and d.
n and m is 1 or zero and p is zero. 14) An insulin derivative or a
pharmaceutically acceptable salt thereof according to any of the
preceding aspects of the general formula
##STR00009## wherein a. Ins represents an insulin comprising a B29
arginine residue and a A22 lysine residue, b. X, X.sup.1 and
X.sup.2 is a fatty diacid substitution, and X is located at said
A22 lysine residue and X.sup.1 is located the N-terminal of the B
chain, and X.sup.2 is not present, c. Z, Z.sup.1 and Z.sup.2 is a
linker between Ins and X, X.sup.1 and X.sup.2, respectively and d.
n and m is 1 or zero and p is zero. 15) An insulin derivative or a
pharmaceutically acceptable salt thereof according to any of the
preceding aspects of the general formula
##STR00010## wherein a. Ins represents an insulin comprising a B29
lysine, b. X, X.sup.1 and X.sup.2 is a fatty diacid substitution,
and X is located at said B29 lysine residue and X.sup.1 is located
at said N-terminal of the A chain, and X.sup.2 is not present c. Z,
Z.sup.1 and Z.sup.2 is a linker between Ins and X, X.sup.1 and
X.sup.2, respectively and d. n and m is 1 or zero and p is zero.
16) An insulin derivative or a pharmaceutically acceptable salt
thereof according to any of the preceding aspects of the general
formula
##STR00011## wherein a. Ins represents an insulin comprising a B29
lysine residue and/or a A22 lysine residue, b. X, X.sup.1 and
X.sup.2 is a fatty diacid substitution, and X is located at said
B29 lysine residue and X.sup.1 is located the N-terminal of the B
chain, and X.sup.2 is not present c. Z, Z.sup.1 and Z.sup.2 is a
linker between Ins and X, X.sup.1 and X.sup.2, respectively and d.
n and m is 1 or zero and p is zero. 17) An insulin derivative or a
pharmaceutically acceptable salt thereof according to any of the
preceding aspects of the general formula
##STR00012## wherein a. Ins represents an insulin comprising a B29
lysine residue and a A22 lysine residue, b. X, X.sup.1 and X.sup.2
is a fatty diacid substitution, and X is located at said B29 lysine
residue and X.sup.1 is located at said A22 lysine residue, and
X.sup.2 is not present c. Z, Z.sup.1 and Z.sup.2 is a linker
between Ins and X, X.sup.1 and X.sup.2, respectively and d. n and m
is 1 or zero and p is zero. 18) An insulin derivative or a
pharmaceutically acceptable salt thereof according to any of the
preceding aspects of the general formula
##STR00013## wherein a. Ins represents an insulin comprising a B29
arginine residue and/or a A22 lysine residue, b. X, X.sup.1 and
X.sup.2 is a fatty diacid substitution, and X is located at said
N-terminal in the A chain and X.sup.1 is located at said N-terminal
in the B chain, and X.sup.2 is not present c. Z, Z.sup.1 and
Z.sup.2 is a linker between Ins and X, X.sup.1 and X.sup.2,
respectively and d. n and m is 1 or zero and p is zero. 19) An
insulin derivative or a pharmaceutically acceptable salt thereof
according to any of the preceding aspects of the general
formula
##STR00014## wherein a. Ins represents an insulin comprising a B29
lysine, b. X, X.sup.1 and X.sup.2 is a fatty diacid substitution,
and X is located at said B29 lysine residue and X.sup.1 is at said
N-terminal of the A chain, and X.sup.2 is located at said
N-terminal in the B chain c. Z, Z.sup.1 and Z.sup.2 is a linker
between Ins and X, X.sup.1 and X.sup.2, respectively and d. n, m
and p is zero or 1. 20) An insulin derivative or a pharmaceutically
acceptable salt thereof according to any of the preceding aspects
of the general formula
##STR00015## wherein a. Ins represents an insulin comprising a B29
arginine residue and a A22 lysine residue, b. X, X.sup.1 and
X.sup.2 is a fatty diacid substitution, and X is located at said
A22 lysine residue and X.sup.1 is at said N-terminal of the A
chain, and X.sup.2 is located at said N-terminal of the B chain c.
Z, Z.sup.1 and Z.sup.2 is a linker between Ins and X, X.sup.1 and
X.sup.2, respectively and d. n, m and p is zero or 1. 21) An
insulin derivative or a pharmaceutically acceptable salt thereof of
the general formula XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein a.
Ins represents an insulin comprising a B29 lysine or B29 arginine
residue and/or a A22 lysine residue, b. X and X.sup.1 is a fatty
diacid substitution, c. Z and Z.sup.1 is a linker between Ins and X
and X.sup.1 respectively, and d. n and m is zero or 1. 22) An
insulin derivative or a pharmaceutically acceptable salt thereof
according to any of the preceding aspects of the general formula
XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein a. Ins represents an
insulin comprising a B29 lysine or B29 arginine residue and/or a
A22 lysine residue, b. X and X.sup.1 is a fatty diacid
substitution, c. Z and Z.sup.1 is a linker between Ins and X and
X.sup.1 respectively, and d. n and m is zero or 1. 23) An insulin
derivative or a pharmaceutically acceptable salt thereof according
to any of the preceding aspects of the general formula
XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein a. Ins represents an
insulin comprising a B29 lysine or B29 arginine residue and/or a
A22 lysine residue, b. X and X.sup.1 is a fatty diacid substitution
and respectively located in a position selected from the group
consisting of: B29 lysine, A22 lysine, N-terminal of the A chain,
N-terminal of the B-chain c. Z and Z.sup.1 is a linker between Ins
and X and X.sup.1 respectively, and d. n and m is zero or 1. 24) An
insulin derivative or a pharmaceutically acceptable salt thereof
according to any of the preceding aspects of the general formula
XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein a. Ins represents an
insulin comprising a B29 arginine residue and a A22 lysine residue,
b. X and X.sup.1 is a fatty diacid substitution fatty diacid
substitution, and X is located at said A22 lysine residue and
X.sup.1 is located the N-terminal of the A chain, c. Z and Z.sup.1
is a linker between Ins and X and X.sup.1 respectively, and d. n
and m is zero or 1. 25) An insulin derivative or a pharmaceutically
acceptable salt thereof according to any of the preceding aspects
of the general formula XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein
a. Ins represents an insulin comprising a B29 arginine residue and
a A22 lysine residue, b. X and X.sup.1 is a fatty diacid
substitution, and X is located at said A22 lysine residue and
X.sup.1 is located the N-terminal of the B chain, c. Z and Z.sup.1
is a linker between Ins and X and X.sup.1 respectively, and d. n
and m is zero or 1. 26) An insulin derivative or a pharmaceutically
acceptable salt thereof according to any of the preceding aspects
of the general formula XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein
a. Ins represents an insulin comprising a B29 lysine residue, b. X
and X.sup.1 is a fatty diacid substitution, and X is located at
said B29 lysine residue and X.sup.1 is located at said N-terminal
of the A chain, c. Z and Z.sup.1 is a linker between Ins and X and
X.sup.1 respectively, and d. n and m is zero or 1. 27) An insulin
derivative or a pharmaceutically acceptable salt thereof according
to any of the preceding aspects of the general formula
XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein a. Ins represents an
insulin comprising a B29 lysine residue, b. X and X.sup.1 is a
fatty diacid substitution, and X is located at said B29 lysine
residue and X.sup.1 is located the N-terminal of the B chain, c. Z
and Z.sup.1 is a linker between Ins and X and X.sup.1 respectively,
and d. n and m is zero or 1. 28) An insulin derivative or a
pharmaceutically acceptable salt thereof according to any of the
preceding aspects of the general formula
XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein a. Ins represents an
insulin comprising a B29 lysine and a A22 lysine residue, b. X and
X.sup.1 is a fatty diacid substitution, and X is located at said
B29 lysine residue and X.sup.1 is located at said A22 lysine
residue, c. Z and Z.sup.1 is a linker between Ins and X and X.sup.1
respectively, and d. n and m is zero or 1. 29) An insulin
derivative or a pharmaceutically acceptable salt thereof according
to any of the preceding aspects of the general formula
XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein a. Ins represents an
insulin comprising a B29 arginine residue, b. X and X.sup.1 is a
fatty diacid substitution, and X is located at said N-terminal of
the A chain and X.sup.1 is located at said N-terminal in the B
chain, c. Z and Z.sup.1 is a linker between Ins and X and X.sup.1
respectively, and d. n and m is zero or 1. 30) An insulin
derivative or a pharmaceutically acceptable salt thereof according
to any of the preceding aspects, wherein said fatty diacid
substitutions comprise 10 to 20 carbon atoms. 31) An insulin
derivative or a pharmaceutically acceptable salt thereof according
to any of the preceding aspects, wherein said fatty acid
substitutions are selected from a group of protracting moieties
selected from Chem. 1 and Chem. 2: HOOC--(CH.sub.2).sub.x--CO--*
Chem 1 HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem 2
Wherein x is an integer from 10 to 20 and y is an integer from 6 to
14. 32) An insulin derivative or a pharmaceutically acceptable salt
thereof according to any of the preceding aspects, wherein said
fatty acid substitutions are selected from a group of protracting
moieties selected from Chem. 1 and Chem. 2:
HOOC--(CH.sub.2).sub.x--CO--* Chem 1
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem 2 Wherein x
is an integer from 14 to 20 and y is an integer from 6 to 10. 33)
An insulin derivative or a pharmaceutically acceptable salt thereof
according to any of the preceding aspects, wherein said fatty acid
substitutions are selected from a group of protracting moieties
selected from Chem. 1 and Chem. 2: HOOC--(CH.sub.2).sub.x--CO--*
Chem 1 HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem 2
Wherein x is an integer from 14 to 18 and y is an integer from 8 to
10. 34) An insulin derivative or a pharmaceutically acceptable salt
thereof according to any of the preceding aspects, wherein said
fatty acid substitutions are selected from a group of protracting
moieties selected from Chem. 1 and Chem. 2:
HOOC--(CH.sub.2).sub.x--CO--* Chem 1
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem 2 Wherein x
is an integer from 14 to 16 and y is an integer from 10 to 12. 35)
An insulin derivative or a pharmaceutically acceptable salt thereof
according to any of the preceding aspects, wherein said fatty acid
substitutions are selected from a group of protracting moieties
selected from Chem. 1 and Chem. 2: HOOC--(CH.sub.2).sub.x--CO--*
Chem 1 HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem 2
Wherein x is an integer from 14 to 20 and y is an integer from 6 to
12. 36) An insulin derivative or a pharmaceutically acceptable salt
thereof according to any of the preceding aspects, wherein said
fatty acid substitutions are selected from a group of protracting
moieties selected from Chem. 1 and Chem. 2:
HOOC--(CH.sub.2).sub.x--CO--* Chem 1
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem 2 Wherein x
is 12, 14, 16, 18 or 20 and y is 6, 8, 10, 12 or 14. 37) An insulin
derivative or a pharmaceutically acceptable salt thereof according
to any of the preceding aspects, wherein said fatty acid
substitutions are selected from a group of protracting moieties
selected from Chem. 1 and Chem. 2: HOOC--(CH.sub.2).sub.x--CO--*
Chem 1 HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem 2
Wherein x is 14, 16, 18 or 20 and y is 8, 10 or 12. 38) An insulin
derivative or a pharmaceutically acceptable salt thereof according
to any of the preceding aspects, wherein said fatty acid
substitutions are selected from a group of protracting moieties
selected from Chem. 1 and Chem. 2: HOOC--(CH.sub.2).sub.x--CO--*
Chem 1 HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem 2
Wherein x is 14, 16, 18 or 20 and y is 6, 8 or 10. 39) An insulin
derivative or a pharmaceutically acceptable salt thereof according
to any of the preceding aspects, wherein said fatty acid
substitutions are selected from a group of protracting moieties
selected from Chem. 1 and Chem. 2: HOOC--(CH.sub.2).sub.x--CO--*
Chem 1 HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem 2
Wherein x is 14, 16 or 18 and y is 8, 10 or 12. 40) An insulin
derivative or a pharmaceutically acceptable salt thereof according
to any of the preceding aspects, wherein said fatty diacid
substitution is attached at an amino acid residue in said insulin,
in a position selected from the group consisting of A1, A22, B1 and
B29. 41) An insulin derivative according to any of the preceding
aspects, wherein said fatty diacid substitution is attached to a
lysine side-chain epsilon-amino group, or the N-terminal of the A
and/or B chain of the insulin, respectively. 42) An insulin
derivative or a pharmaceutically acceptable salt thereof according
to any of the preceding aspects, wherein said fatty diacid
substitutions are attached to amino acid residues of the insulin
via a linker. 43) An insulin derivative or a pharmaceutically
acceptable salt thereof according to any of the preceding aspects,
wherein said linker (Z) comprises one or more linker elements (e).
44) An insulin derivative or a pharmaceutically acceptable salt
thereof according to any of the preceding aspects, wherein said
linker comprises one or more linker elements selected from the
group consisting of: alpha-L-Glu, alpha-D-Glu, gamma-L-Glu,
gamma-D-Glu, alpha-L-Asp, alpha-D-Asp, beta-L-Asp, beta-D-Asp, CPH,
IDA and OEG. 45) An insulin derivative or a pharmaceutically
acceptable salt thereof according to any of the preceding aspects,
wherein said linker comprises two linker elements represented by
the formula: e.sub.1-e.sub.2, wherein a. e.sub.1 is a linker
element selected from the group consisting of alpha-L-Glu,
alpha-D-Glu, gamma-L-Glu, gamma-D-Glu, alpha-L-Asp, alpha-D-Asp,
beta-L-Asp, beta-D-Asp, CPH, IDA and OEG b. e.sub.2 is a linker
element selected from the group consisting of alpha-L-Glu,
alpha-D-Glu, gamma-L-Glu, gamma-D-Glu, alpha-L-Asp, alpha-D-Asp,
beta-L-Asp, beta-D-Asp, CPH, IDA and OEG. 46) An insulin derivative
or a pharmaceutically acceptable salt thereof according to any of
the preceding aspects, wherein said linker comprises three linker
elements represented by the formula: e.sub.1-e.sub.2-e.sub.3,
wherein a. e.sub.1 is a linker element selected from the group
consisting of alpha-L-Glu, alpha-D-Glu, gamma-L-Glu, gamma-D-Glu,
alpha-L-Asp, alpha-D-Asp, beta-L-Asp, beta-D-Asp, CPH, IDA and OEG
b. e.sub.2 is a linker element selected from the group consisting
of alpha-L-Glu, alpha-D-Glu, gamma-L-Glu, gamma-D-Glu, alpha-L-Asp,
alpha-D-Asp, beta-L-Asp, beta-D-Asp, CPH, IDA and OEG c. e.sub.3 is
a linker element selected from the group consisting of alpha-L-Glu,
alpha-D-Glu, gamma-L-Glu, gamma-D-Glu, alpha-L-Asp, alpha-D-Asp,
beta-L-Aps, beta-D-Asp, CPH, IDA and OEG. 47) An insulin derivative
or a pharmaceutically acceptable salt thereof according to any of
the preceding aspects, wherein said linker comprises four linker
elements represented by the formula:
e.sub.1-e.sub.2-e.sub.3-e.sub.4, wherein a. e.sub.1 is a linker
element selected from the group consisting of alpha-L-Glu,
alpha-D-Glu, gamma-L-Glu, gamma-D-Glu, alpha-L-Asp, alpha-D-Asp,
beta-L-Asp, beta-D-Asp, CPH, IDA and OEG b. e.sub.2 is a linker
element selected from the group consisting of alpha-L-Glu,
alpha-D-Glu, gamma-L-Glu, gamma-D-Glu, alpha-L-Asp, alpha-D-Asp,
beta-L-Asp, beta-D-Asp, CPH, IDA and OEG c. e.sub.3 is a linker
element selected from the group consisting of alpha-L-Glu,
alpha-D-Glu, gamma-L-Glu, gamma-D-Glu, alpha-L-Asp, alpha-D-Asp,
beta-L-Aps, beta-D-Asp, CPH, IDA and OEG d. e.sub.4 is a linker
element selected from the group consisting of alpha-L-Glu,
alpha-D-Glu, gamma-L-Glu, gamma-D-Glu, alpha-L-Asp, alpha-D-Asp,
beta-L-Asp, beta-D-Asp, CPH, IDA and OEG. 48) An insulin derivative
or a pharmaceutically acceptable salt thereof according to any of
the preceding aspects, wherein said albumin binding moiety is
located at the N-terminal of said insulin A and/or B-chain and
wherein the linker comprises one or more CPH linker elements. 49)
An insulin derivative according to any of the preceding aspects,
wherein the albumin binding moieties of said insulin derivative are
similar. 50) An insulin derivative or a pharmaceutically acceptable
salt thereof according to any of the preceding aspects, wherein the
insulin derivative is selected from the group consisting of a
derivative of human insulin, a derivative of desB30 human insulin
and a derivative of an insulin analogue. 51) An insulin derivative
or a pharmaceutically acceptable salt thereof according to any of
the preceding aspects, wherein the insulin is selected from the
group consisting of desB30 human insulin, A22K desB30 human
insulin, A14E A22K desB30 human insulin, A14E A22K B25H B29R desB30
human insulin, A14E A22K B25H desB30 human insulin A14E B25H desB27
desB30 human insulin, A14E B25H desB30 human insulin, A14E B16H
desB30 human insulin, A14E B16H B25H desB30 human insulin B28D
human insulin, A22K B29R desB30 human insulin, B3K B28E human
insulin, B28D desB30 human insulin, A22K B29P desB30 human insulin,
B28K B29P human insulin, B28K B29P desB30 human insulin and B3K
B28E desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-gamma-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-gamma-L-glutamyl-OEG-OEG A14E
B16H B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-gamma-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-gamma-L-glutamyl-OEG-OEG A14E
B16H desB27 desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-amino-
methyl-benzyl A14E B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-amino-
methyl-benzyl A14E B25H desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl, B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E desB27 desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-OEG-OEG-4-amino-
methyl-benzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-OEG-OEG
A14E desB27 desB30 human insulin. 52) An insulin derivative or a
pharmaceutically acceptable salt thereof according to any of the
preceding aspects for the use as a medicament in the treatment or
prevention of hyperglycemia including stress induced hyperglycemia,
type 2 diabetes, impaired glucose tolerance, type 1 diabetes,
burns, operation wounds, other diseases or injuries where an
anabolic effect is needed in the treatment, myocardial infarction,
stroke, coronary heart disease, other cardiovascular disorders,
treatment of critically ill diabetic and non-diabetic patients and
polyneuropathy. 53) An insulin derivative or a pharmaceutically
acceptable salt thereof according to any of the preceding aspects
of the general formula XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein
a. Ins represents an insulin comprising a B29 lysine residue, b. X
and X.sup.1 is a fatty diacid substitution, and X is located at
said B29 lysine residue and X.sup.1 is located at said N-terminal
of the A chain and wherein X and X.sup.1 consist of 20 carbon atoms
c. Z and Z.sup.1 is a linker between Ins and X and X.sup.1
respectively, and wherein Z and Z.sup.1 are gGlu-OEG-OEG d. n and m
is 1. 54) An insulin derivative or a pharmaceutically acceptable
salt hereof according to any of the preceding claims, to the extent
possible, selected from the group consisting of
A22N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A22K desB30
human insulin,
A22N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A22K
desB30 human insulin,
A22N.sup..epsilon.-tetradecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-tetradecandioyl-.gamma.-L-glutamyl A22K desB30
human insulin, A22N.sup..epsilon.-octadecandioyl-.gamma.-D-glutamyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-D-glutamyl A22K desB30
human insulin, A22N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A14E A22K B25H
desB30 human insulin,
A22N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
A22K B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A14E B25H
desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl desB30 human
insulin, A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethy-
l-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethy-
l-benzyl A14E B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl
B1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethy-
l-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-be-
nzyl
B1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG4-aminomethyl-be-
nzyl
B1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminometh-
yl-benzyl A14E B25H desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl
B29N.sup..epsilon.octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-N-carboxymethyl-beta-alanyl
B29N.sup..epsilon.-octadecandioyl-N-carboxymethyl-beta-alanyl A14E
B25H desB30 human insulin,
A1N.sup..alpha.octadecandioyl-N-2-carboxyethyl-glycyl
B29N.sup..epsilon.-octadecandioyl-N-2-carboxyethyl-glycyl A14E B25H
desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-N-carboxymethyl-beta-alanyl
A22N.sup..epsilon.-N-octadecandioyl-N-carboxymethyl-beta-alanyl
A22K B29R desB30 human insulin,
A22N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B16H B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E B25H
B29R desB30 human,
A22N.sup..epsilon.-eicosandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-eicosandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
A22K B25H desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B16H B25H desB30 human insulin,
B1N.sup..alpha.-(octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl--
benzyl A14E B25H
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG desB30
human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminom-
ethyl-benzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB27 desB30 human insulin,
A22N.sup..epsilon.-4-carboxyphenoxy-decanoyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-4-carboxyphenoxy-decanoyl-.gamma.-L-glutamyl-OEG-OEG
A14E A22K B25H desB30 human insulin. 55) An insulin derivative or a
pharmaceutically acceptable salt thereof according to any of the
preceding aspects, for the use as a pharmaceutical. 56) An insulin
derivative according to any of the preceding claims, wherein said
insulin derivative is soluble in aqueous solution. 57) A soluble
insulin derivative or a pharmaceutically acceptable salt thereof of
the general formula
##STR00016## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and X.sup.2 is
optional, wherein said fatty diacid substitution comprises 14-20
carbon atoms c. Z, Z.sup.1 and Z.sup.2 is a linker between Ins and
X, X.sup.1 and X.sup.2, respectively and d. n, m and p is zero or
1. 58) A soluble insulin derivative or a pharmaceutically
acceptable salt thereof of the general formula
##STR00017## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and X.sup.2 is
optional, wherein said fatty diacid substitution comprises 14-20
carbon atoms c. Z, Z.sup.1 and Z.sup.2 is a linker between Ins and
X, X.sup.1 and X.sup.2, respectively and d. n, m and p is zero or
1. 59) A soluble insulin derivative or a pharmaceutically
acceptable salt thereof of the general formula
##STR00018## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and X.sup.2 is
optional, wherein said fatty diacid substitution comprises 14-20
carbon atoms c. Z, Z.sup.1 and Z.sup.2 is a linker between Ins and
X, X.sup.1 and X.sup.2, respectively and d. n, m and p is zero or
1. 60) The soluble insulin derivative or pharmaceutically
acceptable salt according to claim 1 for use as a pharmaceutical.
61) The soluble insulin derivative or pharmaceutically acceptable
salt thereof according to any of the preceding claims of the
general formula
##STR00019## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue, b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and respectively
located in a position selected from the group consisting of: B29
lysine, A22 lysine, N-terminal of the A chain, N-terminal of the
B-chain, wherein said fatty diacid substitution comprises 14-20
carbon atoms c. Z, Z.sup.1 and Z.sup.2 is a linker between Ins and
X, X.sup.1 and X.sup.2, respectively and d. n, m and p is zero or
1. 62) The soluble insulin derivative or pharmaceutically
acceptable salt thereof according to any of the preceding claims of
the general formula
##STR00020## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue, b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and respectively
located in a position selected from the group consisting of: A22
lysine, N-terminal of the A chain, N-terminal of the B-chain,
wherein said fatty diacid substitution comprises 14-20 carbon atoms
c. Z, Z.sup.1 and Z.sup.2 is a linker between Ins and X, X.sup.1
and X.sup.2, respectively and d. n, m and p is zero or 1. 63) The
soluble insulin derivative or pharmaceutically acceptable salt
thereof according to any of the preceding claims of the general
formula
##STR00021## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue, b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and respectively
located in a position selected from the group consisting of: B29
lysine, A22 lysine, N-terminal of the B-chain, wherein said fatty
diacid substitution comprises 14-20 carbon atoms c. Z, Z.sup.1 and
Z.sup.2 is a linker between Ins and X, X.sup.1 and X.sup.2,
respectively and d. n, m and p is zero or 1. 64) The soluble
insulin derivative or pharmaceutically acceptable salt thereof
according to any of the preceding claims of the general formula
XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein a. Ins represents an
insulin comprising a B29 lysine or B29 arginine residue and/or a
A22 lysine residue, b. X and X.sup.1 is a fatty diacid
substitution, wherein said fatty diacid substitution comprises
14-20 carbon atoms c. Z and Z.sup.1 is a linker between Ins and X
and X.sup.1 respectively, and d. n and m is zero or 1. 65) The
soluble insulin derivative or pharmaceutically acceptable salt
thereof according to any of the preceding claims of the general
formula XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein a. Ins
represents an insulin comprising a B29 lysine or B29 arginine
residue and/or a A22 lysine residue, b. X and X.sup.1 is a fatty
diacid substitution and respectively located in a position selected
from the group consisting of: B29 lysine, A22 lysine, N-terminal of
the A chain, N-terminal of the B-chain, wherein said fatty diacid
substitution comprises 14-20 carbon atoms 66) The soluble insulin
derivative or pharmaceutically acceptable salt thereof according to
any of the preceding claims of the general formula
##STR00022## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue, b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and respectively
located in a position selected from the group consisting of: B29
lysine, A22 lysine, N-terminal of the A chain, N-terminal of the
B-chain, wherein said fatty diacid substitution comprises 14, 16,
18 or 20 carbon atoms c. Z, Z.sup.1 and Z.sup.2 is a linker between
Ins and X, X.sup.1 and X.sup.2, respectively and d. n, m and p is
zero or 1.
67) The soluble insulin derivative or pharmaceutically acceptable
salt thereof according to any of the preceding claims of the
general formula
##STR00023## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue, b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and respectively
located in a position selected from the group consisting of: A22
lysine, N-terminal of the A chain, N-terminal of the B-chain,
wherein said fatty diacid substitution comprises 14, 16, 18 or 20
carbon atoms c. Z, Z.sup.1 and Z.sup.2 is a linker between Ins and
X, X.sup.1 and X.sup.2, respectively and d. n, m and p is zero or
1. 68) The soluble insulin derivative or pharmaceutically
acceptable salt thereof according to any of the preceding claims of
the general formula
##STR00024## wherein a. Ins represents an insulin comprising a B29
lysine or B29 arginine residue and/or a A22 lysine residue, b. X,
X.sup.1 and X.sup.2 is a fatty diacid substitution and respectively
located in a position selected from the group consisting of: B29
lysine, A22 lysine, N-terminal of the B-chain, wherein said fatty
diacid substitution comprises 14, 16, 18 or 20 carbon atoms c. Z,
Z.sup.1 and Z.sup.2 is a linker between Ins and X, X.sup.1 and
X.sup.2, respectively and d. n, m and p is zero or 1. 69) The
soluble insulin derivative or pharmaceutically acceptable salt
thereof according to any of the preceding claims of the general
formula XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein a. Ins
represents an insulin comprising a B29 lysine or B29 arginine
residue and/or a A22 lysine residue, b. X and X.sup.1 is a fatty
diacid substitution, wherein said fatty diacid substitution
comprises 14, 16, 18 or 20 carbon atoms c. Z and Z.sup.1 is a
linker between Ins and X and X.sup.1 respectively, and d. n and m
is zero or 1. 70) The soluble insulin derivative or
pharmaceutically acceptable salt thereof according to any of the
preceding claims of the general formula
XZ.sub.n-Ins-Z.sup.1.sub.mX.sup.1, wherein a. Ins represents an
insulin comprising a B29 lysine or B29 arginine residue and/or a
A22 lysine residue, b. X and X.sup.1 is a fatty diacid substitution
and respectively located in a position selected from the group
consisting of: B29 lysine, A22 lysine, N-terminal of the A chain,
N-terminal of the B-chain, wherein said fatty diacid substitution
comprises 14, 16, 18 or 20 carbon atoms c. Z and Z.sup.1 is a
linker between Ins and X and X.sup.1 respectively, and d. n and m
is zero or 1. 71) The soluble insulin derivative or
pharmaceutically acceptable salt thereof according to any of the
preceding claims, wherein said fatty acid substitutions are
selected from a group of protracting moieties selected from Chem. 1
and Chem. 2: HOOC--(CH.sub.2).sub.x--CO--* Chem 1
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem 2 Wherein x
is an integer from 10 to 20 and y is an integer from 6 to 14. 72)
The soluble insulin derivative or pharmaceutically acceptable salt
thereof according to any of the preceding claims, wherein said
fatty acid substitutions are selected from a group of protracting
moieties selected from Chem. 1 and Chem. 2:
HOOC--(CH.sub.2).sub.x--CO--* Chem 1
HOOC--C.sub.6H.sub.4--O--(CH.sub.2).sub.y--CO--* Chem 2 Wherein x
is 14, 16 or 18 and y is 8, 10 or 12. 73) The soluble insulin
derivative or pharmaceutically acceptable salt thereof according to
any of the preceding claims, wherein said fatty diacid substitution
is attached at an amino acid residue in said insulin, in a position
selected from the group consisting of A1, A22, B1 and B29. 74) The
soluble insulin derivative or pharmaceutically acceptable salt
thereof according to any of the preceding claims, wherein said
fatty diacid substitutions are attached to amino acid residues of
the insulin via a linker. 75) The soluble insulin derivative or
pharmaceutically acceptable salt thereof according to any of the
preceding claims, wherein said linker comprises one or more linker
elements selected from the group consisting of: alpha-L-Glu,
alpha-D-Glu, gamma-L-Glu, gamma-D-Glu, alpha-L-Asp, alpha-D-Asp,
beta-L-Asp, beta-D-Asp, CPH, IDA and OEG. 76) The soluble insulin
derivative or pharmaceutically acceptable salt thereof according to
any of the preceding claims for the use as a medicament in the
treatment or prevention of hyperglycemia including stress induced
hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1
diabetes, burns, operation wounds, other diseases or injuries where
an anabolic effect is needed in the treatment, myocardial
infarction, stroke, coronary heart disease, other cardiovascular
disorders, treatment of critically ill diabetic and non-diabetic
patients and polyneuropathy. 77) The soluble insulin derivative or
pharmaceutically acceptable salt hereof according to any of the
preceding claims, selected from the group consisting of
A22N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A22K desB30
human insulin,
A22N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A22K
desB30 human insulin,
A22N.sup..epsilon.-tetradecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-tetradecandioyl-.gamma.-L-glutamyl A22K desB30
human insulin, A22N.sup..epsilon.-octadecandioyl-.gamma.-D-glutamyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-D-glutamyl A22K desB30
human insulin, A22N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A14E A22K B25H
desB30 human insulin,
A22N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
A22K B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A14E B25H
desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl desB30 human
insulin, A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethy-
l-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethy-
l-benzyl A14E B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl
B1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethy-
l-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-be-
nzyl
B1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl A14E B25H desB27 desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG4-aminomethyl-be-
nzyl
B1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminometh-
yl-benzyl A14E B25H desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-ben-
zyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl
B29N.sup..epsilon.octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-N-carboxymethyl-beta-alanyl
B29N.sup..epsilon.-octadecandioyl-N-carboxymethyl-beta-alanyl A14E
B25H desB30 human insulin,
A1N.sup..alpha.octadecandioyl-N-2-carboxyethyl-glycyl
B29N.sup..epsilon.-octadecandioyl-N-2-carboxyethyl-glycyl A14E B25H
desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-N-carboxymethyl-beta-alanyl
A22N.sup..epsilon.-N-octadecandioyl-N-carboxymethyl-beta-alanyl
A22K B29R desB30 human insulin,
A22N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin,
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B16H B25H desB30 human insulin,
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E B25H
B29R desB30 human,
A22N.sup..epsilon.-eicosandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-eicosandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
A22K B25H desB30 human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B16H B25H desB30 human insulin,
B1N.sup..alpha.-(octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl--
benzyl A14E B25H
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG desB30
human insulin,
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminom-
ethyl-benzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB27 desB30 human insulin,
A22N.sup..epsilon.-4-carboxyphenoxy-decanoyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-4-carboxyphenoxy-decanoyl-.gamma.-L-glutamyl-OEG-OEG
A14E A22K B25H desB30 human insulin. 78) A method for preparing an
insulin derivative according to any of the above claims comprising
either a step of acylation or alkylation 79) A method for preparing
an insulin derivative according to any of the above aspects. 80) A
method for preparing an insulin derivative according to any of the
above aspects by reductive alkylation and/or acylation of an
insulin. 81) A method for preparing an insulin derivative according
to any of the above aspects by reductive alkylation of an insulin.
82) A method for preparing an insulin derivative according to any
of the above aspects by acylation of an insulin. 83) Use of an
insulin derivative or a pharmaceutically acceptable salt thereof
according to the aspects 1-77 as a medicament. 84) Use of an
insulin derivative or a pharmaceutically acceptable salt thereof
according to the aspects 1-77 for the treatment or prevention of
hyperglycemia including stress induced hyperglycemia, type 2
diabetes, impaired glucose tolerance, type 1 diabetes, burns,
operation wounds, other diseases or injuries where an anabolic
effect is needed in the treatment, myocardial infarction, stroke,
coronary heart disease, other cardiovascular disorders, treatment
of critically ill diabetic and non-diabetic patients and
polyneuropathy. 85) An insulin derivative or a pharmaceutically
acceptable salt thereof according to any of the aspects 1-77,
wherein said fatty diacid substitution X is located in the position
B29 lysine and fatty diacid substitution X.sup.1 is located at the
N-terminal of the A chain of said insulin, wherein X and X.sup.1
consist of 20 carbon atoms and said linkers Z and Z.sup.1 are
gGlu-OEG-OEG. 86) Use of an insulin derivative or a
pharmaceutically acceptable salt thereof according to any of the
aspects 1-77, wherein said fatty diacid substitution X is located
in the position B29 lysine and fatty diacid substitution X.sup.1 is
located at the N-terminal of the A chain of said insulin, wherein X
and X.sup.1 consist of 20 carbon atoms and said linkers Z and
Z.sup.1 are gGlu-OEG-OEG as a medicament. 87) Use of an insulin
derivative or a pharmaceutically acceptable salt thereof according
to any of the aspects 1-77, wherein said fatty diacid substitution
X is located in the position B29 lysine and fatty diacid
substitution X.sup.1 is located at the N-terminal of the A chain of
said insulin, wherein X and X.sup.1 consist of 20 carbon atoms and
said linkers Z and Z.sup.1 are gGlu-OEG-OEG for the treatment or
prevention of hyperglycemia including stress induced hyperglycemia,
type 2 diabetes, impaired glucose tolerance, type 1 diabetes,
burns, operation wounds, other diseases or injuries where an
anabolic effect is needed in the treatment, myocardial infarction,
stroke, coronary heart disease, other cardiovascular disorders,
treatment of critically ill diabetic and non-diabetic patients and
polyneuropathy. 88) An insulin derivative or a pharmaceutically
acceptable salt thereof according to any of the aspects 1-77,
wherein said fatty diacid substitution X is located in the position
B29 lysine and fatty diacid substitution X.sup.1 is located at the
N-terminal of the A chain of said insulin, wherein X and X.sup.1
consist of 20 carbon atoms and said linkers Z and Z.sup.1 are not
gGlu-OEG-OEG. 89) An insulin derivative or a pharmaceutically
acceptable salt thereof according to any of the aspects 1-77,
wherein said fatty diacid substitution X is located in the position
B29 lysine and fatty diacid substitution X.sup.1 is located at the
N-terminal of the A chain of said insulin, and wherein said linkers
Z and Z.sup.1 are gGlu-OEG-OEG and said wherein X and X.sup.1 do
not consist of 20 carbon atoms. 90) An insulin derivative or a
pharmaceutically acceptable salt thereof according to any of the
aspects 1-77, wherein if said fatty diacid substitution X is
located in the position B29 lysine and fatty diacid substitution
X.sup.1 is located at the N-terminal of the A chain of said
insulin, and wherein said linkers Z and Z.sup.1 are gGlu-OEG-OEG,
then said wherein X and X.sup.1 do not consist of 20 carbon atoms.
91) An insulin derivative or a pharmaceutically acceptable salt
thereof according to any of the aspects 1-72, wherein if said fatty
diacid substitution X is located in the position B29 lysine and
fatty diacid substitution X.sup.1 is located at the N-terminal of
the A chain of said insulin, wherein X and X.sup.1 consist of 20
carbon atoms and then said linkers Z and Z.sup.1 are not
gGlu-OEG-OEG
LIST OF ABBREVIATIONS
AcOH, acetic acid Cpm, counts per minute Da, dalton DCM,
dichloromethane DIPEA, N,N-diisopropylethylamine DMSO,
dimethylsulfoxide EDTA, ethylenediamine tetraacetic acid ESI,
Electrospray ionization Fmoc, Fluorenylmethyloxycarbonyl HCCA,
4-hydroxy-.alpha.-cyano-cinnamic acid HEPES,
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPCD,
2-hydroxypropyl-beta-cyclodextrin HPLC, High-performance liquid
chromatography, sometimes referred to as high-pressure liquid
chromatography HSA, human serum albumin LC-MS/LCMS, Liquid
chromatographymass spectrometry IDDM, insulin dependent diabetes
mellitus IEF, Isoelectric focusing NaAc, sodium acetate NIDDM
non-insulin dependent diabetes mellitus NMP, N-methyl-pyrrolidone
MALDI, matrix-assisted laser disorbtion ionisation MRT, Mean
residence time OEG, 8-amino-3,6-dioxaoctanoic acid,
8-amino-3,6-dioxaoctanoyl RP-HPLC, Reversed phase HPLC Rt,
retention time RT Room temperature SPA, scintillation proximity
assay SPA-PVT, scintillation proximity assay polyvinyl toluene bead
T-boc, Di-tert-butyl dicarbonate THF, tetrehydrofurane TFA,
trifluoroacetic acid Tris, tris(hydroxymethyl)aminomethane tRNA,
transfer RNA UV, ultraviolet
EXAMPLES
Example 1
A22N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A22K desB30
human insulin
##STR00025##
A22K desB30 insulin (200 mg, 34 .mu.M) was dissolved in 0.2 M
sodium carbonate, pH 10.5 (2.4 mL) and treated with
hexadecandioyl-.gamma.-succinimidyl-L-glutamate (44 mg, 86 uM,
prepared as described in WO05012347) in acetonitrile (2.4 mL). pH
was measured and adjusted to 10.5 if necessary. After 30 minutes,
the reaction was quenched by addition of 0.2 M methylamine, pH 8
(0.24 mL). pH was adjusted to 5.5 using 1 M HCl and the precipitate
was collected by centrifugation. The product was purified by
RP-HPLC on C18 column using buffer A: 10 mM Tris, 15 mM ammonium
sulfate, pH 7.3 in water/acetonitrile 80/20, buffer B:
water/acetonitrile 20/80, gradient 11% B to 50% B over 60 minutes.
The product was precipitated by adjustment of pH to 5.5 followed by
centrifugation. Alternatively, the product was further purified by
RP-HPLC on C18 column using buffer A: 0.1% trifluoroacetic acid in
water, buffer B: 0.1% trifluoroacetic acid in acetonitrile, with
product pools partially evaporated in vacuo and freeze-dried
providing A22N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A22K desB30
human insulin.
Product LCMS: 1658.4 Da [M+4H].sup.4+.
Calculated for C.sub.301H.sub.458N.sub.68O.sub.88S.sub.6
[M+4H].sup.4+: 1658.5 Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 10 to 90% B over 10
minutes.
Example 2
A22N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A22K
desB30 human insulin
##STR00026##
This compound was prepared in analogy with the compound of example
1 by using octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl
as reagent, prepared as described in WO2010/029159.
Product LCMS: 1817.5 Da [M+4H].sup.4+.
Calculated for C.sub.329H.sub.510N.sub.72O.sub.100S.sub.6
[M+4H].sup.4+: 1817.6 Da.
Example 3
A22N.sup..epsilon.-tetradecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-tetradecandioyl-.gamma.-L-glutamyl A22K desB30
human insulin
##STR00027##
This compound was prepared in analogy with the compound of example
1 by using tetradecandioyl-.gamma.-succinimidyl-L-glutamate as
reagent.
Product LCMS: 1643.9 Da [M+4H].sup.4+.
Calculated for C.sub.297H.sub.450N.sub.68O.sub.88S.sub.6
[M+4H].sup.4+: 1644.4 Da.
Example 4
A22N.sup..epsilon.-octadecandioyl-.gamma.-D-glutamyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-D-glutamyl A22K desB30
human insulin
##STR00028##
This compound was prepared in analogy with the compound of example
1 by using
tert-butyl-octadecandioyl-.gamma.-succinimidyl-D-glutamate-tert-but-
yl as reagent, prepared as described in WO05012347. The tert-butyl
protecting groups were removed by treatment of the crude product
with ice-cooled 95% trifluoroacetic acid/water for 45 minutes.
Product LCMS: 1672.2 Da [M+4H].sup.4+.
Calculated for C.sub.305H.sub.466N.sub.68O.sub.88S.sub.6
[M+4H].sup.4+: 1672.5 Da.
Example 5
A22N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A14E A22K B25H
desB30 human insulin
##STR00029##
This compound was prepared in analogy with the compound of example
1 by using hexadecandioyl-.gamma.-succinimidyl-L-glutamate and A14E
A22K B25H desB30 human insulin.
Product LCMS: 1647.3 Da [M+4H].sup.4+.
Calculated for C.sub.294H.sub.454N.sub.70O.sub.89S.sub.6
[M+4H].sup.4+: 1647.4 Da.
Example 6
A22N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
A22K B25H desB30 human insulin
##STR00030##
This compound was prepared in analogy with the compound of example
1 by using octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl
and A14E A22K B25H desB30 human insulin.
Product LCMS: 1806.6 Da [M+4H].sup.4+.
Calculated for C.sub.322H.sub.506N.sub.74O.sub.101S.sub.6
[M+4H].sup.4+: 1806.6 Da.
Example 7
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl A14E B25H
desB30 human insulin
##STR00031##
This compound was prepared in analogy with the compound of example
1 in 1:1 acetonitrile/0.2 M sodium carbonate, pH 9.0, by using
hexadecandioyl-.gamma.-succinimidyl-L-glutamate and A14E B25H
desB30 human insulin.
Product LCMS: 1615.3 Da [M+4H].sup.4+.
Calculated for C.sub.288H.sub.442N.sub.68O.sub.88S.sub.6
[M+4H].sup.4+: 1615.4 Da.
Affinity for HSA column, Rt (min): 8.0 minutes, the value for
mono-substituted insulin C (see example 29 and table 1) 6.4
mins
Example 8
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl desB30 human
insulin
##STR00032##
This compound was prepared in analogy with the compound of example
1 in 1:1 acetonitrile/0.2 M sodium carbonate, pH 9.0, by using
hexadecandioyl-.gamma.-succinimidyl-L-glutamate and desB30 human
insulin.
Product LCMS: 1626.4 Da [M+4H].sup.4+.
Calculated for C.sub.295H.sub.446N.sub.68O.sub.87S.sub.6
[M+4H].sup.4+: 1626.4 Da.
Example 9
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin
##STR00033##
This compound was prepared in analogy with the compound of example
1 in 1:1 acetonitrile/0.2 M sodium carbonate, pH 9.0, by using
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl and A14E
B25H desB30 human insulin.
Product LCMS: 1774.7 Da [M+4H].sup.4+.
Calculated for C.sub.316H.sub.494N.sub.72O.sub.100S.sub.6
[M+4H].sup.4+: 1774.6 Da.
Example 10
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-be-
nzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminometh-
yl-benzyl A14E B25H desB27 desB30 human insulin
##STR00034##
Preparation of
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyde
t-Butyl-N-(4-formylbenzyl)carbamate (100 mg) was treated with
TFA/DCM (1:1) for 1 h. The mixture was concentrated in vacuo and
co-concentrated with toluene twice. The residue was dissolved in
THF (2.5 mL) and a solution of
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimydyl ester (320
mg, prepared as described previously in WO2009/083549) in THF (5
mL) was added. DIPEA (0.5 mL) was added slowly. After 130 min, the
mixture was concentrated in vacuo. The residue was dissolved in
EtOAc and 1N HCl. The organic layer was extracted with 1N HCl and
brine. The organic layer was dried (Na.sub.2SO.sub.4) and
concentrated in vacuo to give the title compound as a white solid,
which was used without further purification. Yield 234 mg
(72%).
LCMS: Theoretical mass: 851.0 Found: 851.5 (M+1).
A zinc precipitate of A14E B25H desB27 desB30 human insulin
corresponding to approximately 400 mg insulin was dissolved in
water (32 mL) and EDTA (40 mg) was added. The mixture was left at
RT or 1 h. pH was lowered to 4.8 with 10% AcOH.
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyde
(92 mg) dissolved in 1M NaAc (3.0 mL, heating under tap water) was
added. After stirring for 10 min, 1M NaCNBH3 in water (0.715 mL)
was added to give a 20 mM solution. Within a minute, the mixture
became unclear and a sticky precipitate appeared. After 1 h more
aldehyde (36 mg) and THF (3 mL) was added. After 40 minutes pH was
lowered to 3.1 with AcOH and some Acetonitrile was added. The
mixture was lyophilised. The product was purified by RP-HPLC on a
C18 column using A: 0.1% trifluoroacetic acid in water, buffer B:
0.1% trifluoroacetic acid in acetonitrile. Gradient 20% B to 60% B
over 45 minutes. Product pools were partially evaporated in vacuo
and freeze-dried providing
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl A14E B25H desB27 desB30 human insulin
MALDI: (matrix, HCCA); m/z: 7233.5 Da, calculated for
C.sub.328H.sub.505N.sub.73O.sub.98S.sub.6: 7231.5 Da.
Example 11
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-be-
nzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminometh-
yl-benzyl A14E B25H desB30 human insulin
##STR00035##
A14E B25H desB30 human insulin (300 mg) was dissolved in 1.0 M
NaAc, pH 5.0 (3.2 mL). A solution of aldehyde
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyde
(79 mg) in 1.0 M NaAc, pH 5.0 (3.0 mL, heating under tap water) was
added. The mixture gets unclear. After stirring for 5 minutes 1M
NaCNBH.sub.3 (135 .mu.L) was added to give a 20 mM solution. pH is
4.9 and the reaction mixture appeared unclear. After 40 minutes pH
was lowered to 3.1 with AcOH and some Acetonitrile was added. The
mixture was lyophilised. The product was purified by RP-HPLC on a
C18 column using A: 0.1% trifluoroacetic acid in water, buffer B:
0.1% trifluoroacetic acid in acetonitrile. Gradient 20% B to 60% B
over 45 minutes. Product pools were partially evaporated in vacuo
and freeze-dried providing
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl A14E B25H desB30 human insulin
MALDI: (matrix, HCCA); m/z: 7334.5 Da, calculated for
C.sub.332H.sub.512N.sub.74O.sub.100S.sub.6: 7332.6 Da.
Example 12
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-benz-
yl
B1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-
-benzyl A14E B25H desB27 desB30 human insulin
##STR00036##
A zinc precipitate of A14E B25H desB27 desB30 human insulin
corresponding to approximately 400 mg insulin was dissolved in
water (48 mL) and EDTA (40 mg) was added. The mixture was left at
RT for 45 min. pH was lowered to 5.0 with 10% AcOH.
Hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyde
(90 mg, prepared similarly
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyde)
dissolved in 1M NaAc (3.0 mL, heating under tap water) was added.
After stirring for 7 min, 1M NaCNBH.sub.3 in water (1.08 mL) was
added to give a 20 mM solution. Within a minute, the mixture got
unclear, after a while a sticky precipitate appeared. After 45
minutes pH was lowered to 3.1 with AcOH and some acetonitrile was
added. The mixture was lyophilised. The product was purified by
RP-HPLC on a C18 column using A: 0.1% trifluoroacetic acid in
water, buffer B: 0.1% trifluoroacetic acid in acetonitrile.
Gradient 20% B to 60% B over 45 minutes pH. Product pools were
partially evaporated in vacuo and freeze-dried providing
A1N.sup..alpha.-Hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl A14E B25H desB27 desB30 human insulin
MALDI: (matrix, HCCA); m/z: 7178.9 Da, Calculated for
C.sub.324H.sub.497N.sub.73O.sub.98S.sub.6: 7175.4 Da.
Example 13
A1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminome-
thyl-benzyl A14E B25H desB27 desB30 human insulin
##STR00037##
A zinc precipitate of A14E B25H desB27 desB30 human insulin
corresponding to approximately 100 mg insulin was dissolved in
water (16 mL) and EDTA (10 mg) was added. The mixture was left at
RT for 1 h. pH was lowered to 4.8 with 10% AcOH.
Tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyde
(36 mg, prepared similarly as
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyde)
dissolved in 1M NaAc (0.75 mL, heating under tap water) was added.
After stirring for 10 min, 1M NaCNBH.sub.3 in water (0.35 mL) was
added to give a 20 mM solution. Within a minute, the mixture got
unclear after a while a sticky precipitate appeared. After 1 h more
aldehyde (36 mg) and THF (3 mL) was added. After 4 h the mixture
was stored at 5.degree. C. over night. The product was purified by
RP-HPLC on a C18 column using A: 0.1% trifluoroacetic acid in
water, buffer B: 0.1% trifluoroacetic acid in acetonitrile.
Gradient 20% B to 60% B over 45 minutes. Product pools were
partially evaporated in vacuo and freeze-dried providing
A1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl--
benzyl
B1N.sup..alpha.-tetradecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminom-
ethyl-benzyl A14E B25H desB27 desB30 human insulin
LCMS: 1780.6 Da [M+4H].sup.4+. Calculated for
C.sub.320H.sub.489N.sub.73O.sub.98S.sub.6 [M+4H].sup.4+: 1780.8
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 10 to 90% B over 10
minutes.
MALDI: (matrix, HCCA); m/z: 7118.7 Da, calculated: 7119.3 Da.
Example 14
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG4-aminomethyl-ben-
zyl
B1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethy-
l-benzyl A14E B25H desB30 human insulin
##STR00038##
A14E B25H desB30 human insulin (300 mg) was dissolved in 1.0 M
NaAc, pH 5.0 (3.2 mL). A solution of
hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethyl-benzaldehyde
(79 mg) in 1.0 M NaAc, pH 5.0 (3.0 mL, heating under tap water) was
added. The mixture gets unclear. After stirring for 5 minutes 1M
NaCNBH.sub.3 (135 .mu.L) was added to give a 20 mM solution. pH is
4.9 and the reaction mixture is unclear but chaning. After 40
minutes pH was lowered to 3.1 with AcOH and some acetonitrile was
added. The mixture was lyophilised. The product was purified by
RP-HPLC on a C18 column using A: 0.1% trifluoroacetic acid in
water, buffer B: 0.1% trifluoroacetic acid in acetonitrile.
Gradient 20% B to 60% B over 45 minutes. Product pools were
partially evaporated in vacuo and freeze-dried providing
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomet-
hyl-benzyl A14E B25H desB30 human insulin
MALDI: (matrix, HCCA); m/z: 7279.4 Da, calculated for
C.sub.328H.sub.504N.sub.74O.sub.100S.sub.6: 7276.5 Da.
Example 15
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-be-
nzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B25H desB30 human insulin
##STR00039##
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin, 300 mg, prepared as described in WO
2010/029159 was dissolved in 2M AcOH/NMP 9:1 (9 mL). pH was
adjusted from 2.9 to 3.5 with 1N NaOH.
Octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzalde-
hyde (44 mg) in NMP (0.5 mL) was added. The mixture turned unclear,
pH 3.77. After stirring for 15 minutes 1M 2-picoline borane complex
in NMP/1M NaAc (0.45 mL) was added. pH is 3.77. The mixture was
stirred at Rt. Sticky material gathered on top of the mixture.
After 7 minutes the mixture was diluted with 1M AcOH (4.7 mL, to
dilute the boride). After 1 h pH was lowered to 3.0 with AcOH to
give a solution, which was purified by RP-HPLC on a C18 column
using A: 0.1% trifluoroacetic acid in water, buffer B: 0.1%
trifluoroacetic acid in acetonitrile. Gradient 20% B to 60% B over
45 minutes. Product pools were partially evaporated in vacuo and
freeze-dried providing
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B25H desB30 human insulin
LCMS: 1804.8 Da [M+4H].sup.4+. Calculated for
C.sub.324H.sub.503N.sub.73O.sub.100S.sub.6 [M+4H].sup.4+: 1804.4
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 10 to 90% B over 10
minutes.
MALDI: (matrix, HCCA); m/z: 7215.9 Da, calculated for
C.sub.324H.sub.503N.sub.73O.sub.100S.sub.6: 7213.4 Da.
Example 16
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-be-
nzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminometh-
yl-benzyl B29N.sup..epsilon.
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E B25H desB30 human
insulin
##STR00040##
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin (300 mg) was dissolved in 2 M AcOH/NMP
9:1 (9 mL). pH was adjusted from 2.9 to 3.5 with 1N NaOH, a sticky
suspension was obtained.
Octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzald-
ehyde (44 mg) in NMP (0.5 mL) was added. The mixture turned
unclear, pH is 3.77. After stirring for 15 minutes 1M 2-picoline
borane complex in NMP/1M NaAc (0.45 mL) was added. pH is 3.77. The
mixture was stirred at Rt. Sticky material gathered on top of the
mixture. After 7 minutes the mixture was diluted with 1M AcOH (4.7
mL, to dilute the boride). After 1 h pH was lowered to 3.0 with
AcOH to give a solution, which was purified by RP-HPLC on a C18
column using A: 0.1% trifluoroacetic acid in water, buffer B: 0.1%
trifluoroacetic acid in acetonitrile. Gradient 20% B to 60% B over
45 minutes. Product pools were partially evaporated in vacuo and
freeze-dried providing
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-aminomethy-
l-benzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin.
This example illustrates that a reaction of this type gives rise to
B1 N-alkylation as main product.
LCMS: 1610.9 Da [M+5H].sup.5+. Calculated for
C.sub.367H.sub.573N.sub.77O.sub.112S.sub.6 [M+5H].sup.5+: 1610.7
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 10 to 90% B over 10
minutes.
MALDI: (matrix, HCCA); m/z: 8050.7 Da, calculated for
C.sub.367H.sub.573N.sub.77O.sub.112S.sub.6: 8048.5 Da.
Example 17
A1N.sup..alpha.-octadecandioyl-N-carboxymethyl-beta-alanyl
B29N.sup..epsilon.-octadecandioyl-N-carboxymethyl-beta-alanyl A14E
B25H desB30 human insulin
##STR00041##
A14E B25H desB30 human insulin (200 mg) was dissolved in 100 mM
aqueous Na.sub.2CO.sub.3 (4.5 mL), and pH adjusted to 10.1 with 1 N
NaOH. tert-Butyl
octadecandioyl-N-(tert-butoxycarbonylmethyl)-beta-alanyl-OSu (28
mg) (prepared as described in WO 2005/012347) was dissolved in THF
(2.25 mL) and added to the insulin solution. Some precipitation was
observed and more THF (0.75 mL) was added. Ph was 10.7. After 34
minutes more tert-butyl
octadecandioyl-N-(tert-butoxycarbonylmethyl)-beta-alanyl-OSu (14
mg) was added. After 57 minutes water was added and pH was adjusted
to 5.1 with 1N HCl. The precipitate was spinned down and
lyophilised. The solid was dissolved in ice-cold 95%
trifluoroacetic acid (containing 5% water) and kept on ice for 40
minutes. The mixture was concentrated in vacuo and re-evaporated
from dichloromethane. The residue was purified by RP-HPLC on a C18
column using A: 0.1% trifluoroacetic acid in water, buffer B: 0.1%
trifluoroacetic acid in acetonitrile. Gradient 20%1B to 55% B over
75 minutes. Product pools were partially evaporated in vacuo and
freeze-dried providing
A1N.sup..alpha.-octadecandioyl-N-carboxymethyl-beta-alanyl
B29N.sup..epsilon.-octadecandioyl-N-carboxymethyl-beta-alanyl A14E
B25H desB30 human insulin
LCMS: 1629.0 Da [M+4H].sup.4+. Calculated for
C.sub.292H.sub.450N.sub.68O.sub.88S.sub.6 [M+4H].sup.4+: 1629.4
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 10 to 90% B over 10
minutes.
MALDI: (matrix, HCCA); m/z: 6511.0 Da, calculated for
C.sub.292H.sub.450N.sub.68O.sub.88S.sub.6: 6513.6 Da.
Example 18
A1N.sup..alpha.octadecandioyl-N-2-carboxyethyl-glycyl
B29N.sup..epsilon.-octadecandioyl-N-2-carboxyethyl-glycyl A14E B25H
desB30 human insulin
##STR00042##
A14E B25H desB30 human insulin (100 mg) was dissolved in DMSO (1.0
mL) and triethylamine (0.05 mL) was added. tert-Butyl
octadecandioyl-N-(2-(tert-butoxycarbonyl)ethyl)-Gly-OSu (46 mg)
(prepared as described in WO 2005/012347) dissolved in
acetonitrile/THF 1:1 (2.25 mL) was added. After stirring for 30
minutes at room temperature more tert-Butyl
octadecandioyl-N-(2-(tert-butoxycarbonyl)ethyl)-Gly-OSu (46 mg)
dissolved in acetonitrile/THF 1:1 (2.25 mL) was added. After 75
minutes water was added (5 mL) and pH was adjusted to 5.3 with 1N
HCl. The precipitate was spinned down and lyophilised. The dry
mixture wad treated with 0.1N NaOH at pH 12 on an ice bath for 45
min. pH was readjusted to 5.3 and the precipitate was spinned down
and lyophilized. The solid was dissolved in ice-cold 95%
trifluoroacetic acid (containing 5% water) and kept on ice for 45
minutes. The mixture was concentrated in vacuo and re-evaporated
from dichloromethane. The residue was purified by RP-HPLC on a C18
column using A: 0.1% trifluoroacetic acid in water, buffer B: 0.1%
trifluoroacetic acid in acetonitrile. Gradient 25% 1B to 70% B over
60 minutes. Product pools were partially evaporated in vacuo and
freeze-dried providing
A1N.sup..alpha.-octadecandioyl-N-2-carboxyethyl-glycyl
B29N.sup..epsilon.-octadecandioyl-N-2-carboxyethyl-glycyl A14E B25H
desB30 human insulin
LCMS: 1628.7 Da [M+4H].sup.4+. Calculated for
C.sub.292H.sub.450N.sub.68O.sub.88S.sub.6 [M+4H].sup.4+: 1629.4
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 10 to 90% B over 10
minutes.
MALDI: (matrix, HCCA); m/z: 6511.1 Da, calculated for
C.sub.292H.sub.450N.sub.68O.sub.88S.sub.6. 6513.6 Da.
Example 19
A1N.sup..alpha.-octadecandioyl-N-carboxymethyl-beta-alanyl
A22N.sup..epsilon.-N-octadecandioyl-N-carboxymethyl-beta-alanyl
A22K B29R desB30 human insulin
##STR00043##
A22K B29R desB30 human insulin (200 mg) was dissolved in 100 mM
aqueous Na.sub.2CO.sub.3 (4.5 mL), and pH adjusted to 10.1 with 1 N
NaOH. tert-Butyl
octadecandioyl-N-(tert-butoxycarbonylmethyl)-.beta.Ala-OSu (27 mg)
(prepared as described in WO 2005/012347) was dissolved in
acetonitrile/THF 1.2 (2.25 mL) and added to the insulin solution.
Some precipitation was observed and more THF (1.0 mL) was added to
give a clear solution. Ph was 10.7. After 57 minutes more
tert-butyl
octadecandioyl-N-(tert-butoxycarbonylmethyl)-.beta.Ala-OSu (14 mg)
dissolved in THF (1.1 mL) was added. After 90 min, water (4.5 mL)
was added and pH was adjusted to 5.5 with 1N HCl. The precipitate
was spinned down and lyophilised. The solid was dissolved in
ice-cold 95% trifluoroacetic acid (containing 5% water) and kept on
ice for 25 minutes. The mixture was concentrated in vacuo. The
residue was purified by RP-HPLC on a C18 column using A: 0.1%
trifluoroacetic acid in water, buffer B: 0.1% trifluoroacetic acid
in acetonitrile. Gradient 10% B to 55% B over 75 minutes. Product
pools were partially evaporated in vacuo and freeze-dried providing
A1N.sup..alpha.-octadecandioyl-N-carboxymethylybeta-alanyl)
A22N.sup..epsilon.-octadecandioyl-N-carboxymethyl-beta-alanyl A22K
B29R desB30 human insulin.
LCMS: 1679.0 Da [M+4H].sup.4+. Calculated for
C.sub.305H.sub.466N.sub.70O.sub.88S.sub.6 [M+4H].sup.4+: 1679.5
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 10 to 90% B over 10
minutes.
MALDI: (matrix, HCCA); m/z: 6709.4 Da, calculated for
C.sub.305H.sub.466N.sub.70O.sub.88S.sub.6: 6713.9 Da.
Example 20
A22N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin
##STR00044##
A14E A22K B25H desB30 human insulin (0.5 g, 86 .mu.mol) was
dissolved in 200 mM Na.sub.2CO.sub.3 (5 mL) and pH was adjusted to
11 with 1N NaOH. Then,
hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl (150 mg, 186
.mu.mol) dissolved in NMP (0.5 mL) and acetonitrile (0.1 mL) were
added to the insulin solution and 1N NaOH was added to keep pH at
11. The reaction was stirred for 5 minutes and the progress of
reaction was monitored by LCMS.
Octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl (150 mg, 186
.mu.mol) dissolved in NMP (0.5 mL) and acetonitrile (0.1 mL) were
added further 2 times (using the above protocol) before the target
product was formed monitored by LCMS. The product was purified by
RP-HPLC on C18 column using buffer A: 0.1% TFA in water, Buffer B:
0.1% TFA in acetonitrile and the gradient 25-50% acetonitrile over
60 minutes with a flow of 25 mL/min. The product was purified 2
times. Column: Phenomenex, Gemini, 5.mu., C18, 110 .ANG.,
250.times.30 cm. The pure fractions were then pooled and freeze
dried.
LCMS: 1791.7 Da [M+4H].sup.4+. Calculated for
C.sub.318H.sub.498N.sub.74O.sub.101S.sub.6 [M+4H].sup.4+: 1790.5
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 5 to 90% B over 4
minutes.
Example 21
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B25H desB30 human insulin
##STR00045##
A14E B25H desB30 human insulin (500 mg, 88 .mu.mol) was dissolved
in 200 mM Na.sub.2CO.sub.3 (5 mL) and pH was adjusted to 11 with 1N
NaOH. Then, hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl
(150 mg, 186 .mu.mol) dissolved in NMP (0.5 mL) and acetonitrile
(0.1 mL) were added to the insulin solution and 1N NaOH was added
to keep pH at 11. The reaction was stirred for 5 minutes and the
progress of reaction was monitored by LCMS. The linker,
hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl (150 mg, 186
.mu.mol) dissolved in NMP (0.5 mL) and acetonitrile (0.1 mL) were
added further 3 times (using the above protocol) before the target
product was the major product monitored by LCMS. The product was
purified by RP-HPLC on C18 column using buffer A: 0.1% TFA in
water, Buffer B: 0.1% TFA in acetonitrile and the gradient 30-45%
acetonitrile over 60 minutes with a flow of 25 mL/min. Column:
Phenomenex, Gemini, 5.mu., C18, 110 .ANG., 250.times.30 cm. The
product was purified 2 times. The pure fractions were then pooled
and freeze dried.
LCMS: 1760.3 Da [M+4H].sup.4+. Calculated for
C.sub.312H.sub.486N.sub.72O.sub.100S.sub.6 [M+4H].sup.4+: 1758.5
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 5 to 90% B over 4
minutes.
Example 22
A1N.sup..alpha.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B16H B25H desB30 human insulin
##STR00046##
A14E B16H B25H, desB30 human insulin (1 g, 177 .mu.mol) was
dissolved in 200 mM Na2CO3 (12.5 mL) and pH was adjusted to 11 with
addition of 1N NaOH. Then,
hexadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl (400 mg, 499
.mu.mol) dissolved in NMP (1.0 mL) and acetonitrile (0.2 mL) were
added to the insulin solution over 10 min. 1N NaOH was added
additional to keep pH at 11.0 during the reaction. The reaction
mixture was stirred for 30 minutes at room temperature and progress
of reaction was monitored by LCMS and the product was purified by
RP-HPLC on C18 column using buffer A: 0.1% TFA in water, Buffer B:
0.1% TFA in acetonitrile and the gradient 25-40% acetonitrile over
60 minutes with a flow of 25 mL/min. The pure fractions were then
pooled and freeze dried.
LCMS: 1402.9 Da [M+5H].sup.5+. Calculated for
C.sub.309H.sub.484N.sub.74O.sub.99S.sub.6 [M+5H].sup.5+: 1403.0
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 5 to 90% B over 4
minutes.
Example 23
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E B25H
B29R desB30 human insulin
##STR00047##
A14E B25H B29R desB30 human insulin (0.35 g, 61 .mu.mol) was
dissolved in H2O (3 mL) and pH was adjusted to 11. Then,
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl (100 mg, 120
.mu.mol) dissolved in NMP (0.4 mL) and acetonitrile (0.1 mL) were
added to the insulin solution and 1N NaOH was added to keep pH at
11. The reaction was stirred for 5 minutes and the progress of
reaction was monitored by LCMS.
Octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl (150 mg, 120
.mu.mol) dissolved in NMP (0.4 mL) and acetonitrile (0.1 mL) were
added further and the progress of the formed major product was
monitored by LCMS. The product was purified 2 times by RP-HPLC on
C18 column using buffer A: 0.1% TFA in water, Buffer B: 0.1% TFA in
acetonitrile and the gradients; 30-45% acetonitrile over 60 minutes
and 10-40% acetonitrile over 60 minutes with a flow of 25 mL/min.
The pure fractions were then pooled and freeze dried.
LCMS: 1189.9.88 Da [M+6H].sup.6+. Calculated for
C.sub.316H.sub.494N.sub.74O.sub.100S.sub.6 [M+6H].sup.6+: 1188
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 5 to 90% B over 4
minutes.
Example 24
A22N.sup..epsilon.-eicosandioyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-eicosandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
A22K B25H desB30 human insulin
##STR00048##
A14E A22K B25H desB30 human insulin (450 mg, 77 .mu.mol) was
dissolved in 200 mM Na.sub.2CO.sub.3 (5 mL) and pH was adjusted to
11 with 1N NaOH. Then,
eicosandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl (150 mg, 174
.mu.mol) dissolved in NMP (0.5 mL) and acetonitrile (0.1 mL) were
added to the insulin solution and 1N NaOH was added to keep pH at
11. The reaction was stirred for 5 min. Then, the progress of
reaction was monitored by LCMS
Eicosandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl (150 mg, 174
.mu.mol) dissolved in NMP (0.5 mL) and acetonitrile (0.1 mL) was
added further 2 times (using the above protocol) before the target
product was formed monitored by LCMS. The product was purified 2
times by RP-HPLC on C18 column using 1) buffer A: 0.1% TFA in
water, Buffer B: 0.1% TFA in acetonitrile and the gradient; 25-50%
acetonitrile over 40 minutes with a flow of 25 mL/min 2) buffer A:
10 mM Tris, 15 mM ammonium sulfate, pH 7.3 in water/acetonitrile
80/20, buffer B: water/acetonitrile 20/80 and the gradient; 10-60%
buffer B over 60 minutes with a flow of 25 mL/min. The pure
fractions were then pooled and freeze dried.
LCMS: 1820.1 Da [M+4H].sup.4+. Calculated for
C.sub.326H.sub.514N.sub.74O.sub.101S.sub.6 [M+4H].sup.4+: 1820.5
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 5 to 90% B over 4
minutes.
Example 25
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-be-
nzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B16H B25H desB30 human insulin
##STR00049##
Octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyde
(100 mg, 117 .mu.mol) was dissolved in 2 mL 25 mM HEPES (2 mL) by
heating (tap water) and 20% HPCD (500 .mu.L) was then added to give
a unclear but homogeneous solution. To
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E
B16H B25H desB30 human insulin (250 mg, 39.8 .mu.mol) was added 25
mM HEPES (5 mL, pH 5.6) and pH was adjusted to 5.0 with 1 N HCl.
The above aldehyde solution (2.5 mL) was added to insulin and the
solution became unclear. After 5 min, 1M NaCNBH.sub.3 in MeOH (165
.mu.L) was added and the progress of the reaction was monitored
both by LCMS and UPLC.
The product was purified by RP-HPLC on C18 column using buffer A:
0.1% TFA in water, Buffer B: 0.1% TFA in acetonitrile and the
gradient; 30-55% acetonitrile over 40 minutes with a flow of 25
mL/min. The pure fractions were then pooled and freeze dried.
LCMS: 1438.3 Da [M+5H].sup.5+. Calculated for
C.sub.321H.sub.501N.sub.75O.sub.99S.sub.6 [M+5H].sup.5+: 1438.4
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 5 to 90% B over 4
minutes. Amino acid sequencing of the peptide verified that
C18diacid-.gamma.-LGlu-OEG-OEG-aminomethyl-benzyl was attached to
B1 (alkylation).
Example 26
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-be-
nzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B25H desB30 human insulin
##STR00050##
Octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyde
(100 mg, 117 mmol) was dissolved in 25 mM HEPES (2 mL) by heating
(tap water). 20% HPCD (500 .mu.L) was then added to give a unclear
but homogeneous solution.
To B29N.sup.e-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG A14E B25H
desB30 human insulin (250 mg, 39 .mu.mol) was added 25 mM HEPES (5
mL, pH 5.6) and pH was adjusted to 5.0 with 1 N HCl). The aldehyde
solution (2.5 mL) was added to insulin and the solution became
unclear. After 5 minutes, 1M NaCNBH.sub.3 in MeOH (165 .mu.L) was
added. The progress of the reaction was monitored both by LCMS and
uplc. The product was purified by RP-HPLC on C18 column using
buffer A: 0.1% TFA in water, Buffer B: 0.1% TFA in acetonitrile and
the gradient; 30-55% acetonitrile over 40 minutes with a flow of 25
mL/min. Kolonne: Phenomenex, Gemini, 5.mu., C18, 110 .ANG.,
250.times.30 cm. The pure fractions were then pooled and freeze
dried.
LCMS: 1802.4 Da [M+4H].sup.4+. Calculated for
C.sub.324H.sub.503N.sub.73O.sub.100S.sub.6 [M+4H].sup.4+: 1804
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 5 to 90% B over 4
minutes. Amino acid sequencing of the peptide verified that
C18diacid-.gamma.-LGlu-OEG-OEG-4-aminomethyl-benzyl was attached to
B1 (alkylation).
Example 27
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-be-
nzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B25H desB27 desB30 human insulin
##STR00051##
Octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyde
(140 mg, 163 .mu.mol) was dissolved in 25 mM HEPES (2.8 mL) by
heating (tap water). 20% HPCD (700 .mu.L) was added to give a
solution which was unclear but homogeneous.
To B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E B25H desB27 desB30 human insulin (350 mg, 55 .mu.mol) was
added 25 mM HEPES (7 mL, pH 5.6) and pH was adjusted to 5.5 with 1
N HCl. Aldehyde solution (3.5 mL) was added and the solution was
unclear. A few minutes later 1M NaCNBH.sub.3 in MeOH (230 .mu.L)
was added. After 30 minutes was the progress of the reaction
monitored by LCMS and the desired product was then formed. 1N HCl
was added to acidified the reaction mixture before preparative HPLC
purification. The product was purified by RP-HPLC on C18 column
using buffer A: 0.1% TFA in water, Buffer B: 0.1% TFA in
acetonitrile and the gradient; 25-65% acetonitrile over 40 minutes
with a flow of 25 mL/min. The pure fractions were then pooled and
freeze dried.
LCMS: 1423.4 Da [M+5H].sup.5+. Calculated for
C.sub.320H.sub.496N.sub.72O.sub.98S.sub.6 [M+5H].sup.5+: 1423.5
Da.
LCMS buffer A: 0.1% trifluoroacetic acid in water; buffer B: 0.1%
trifluoroacetic acid in acetonitrile, gradient 5 to 90% B over 4
minutes. Amino acid sequencing of the peptide verified that
C18diacid-.gamma.-LGlu-OEG-OEG-4-aminomethyl-benzyl was attached to
B1 (alkylation).
Example 28
A22N.sup..epsilon.-4-carboxyphenoxy-decanoyl-.gamma.-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-4-carboxyphenoxy-decanoyl-.gamma.-L-glutamyl-OEG-OEG
A14E A22K B25H desB30 human insulin
##STR00052##
This compound was prepared in analogy with the compound of example
4 by using
4-tert-butyl-carboxyphenoxy-decanoyl-.gamma.-L-glutamyl-.alpha.-ter-
t-butyl-OEG-OEG-succinimidyl (prepared in analogue with the
description in WO06082204) and A14E B25H A22K desB30 human
insulin.
Product LCMS: 1806.5 Da [M+4H].sup.4+.
Calculated for C.sub.320H.sub.486N.sub.74O.sub.103S.sub.6
[M+4H].sup.4+: 1803.6 Da.
Example 29
Human Insulin Receptor Affinity, Albumin Affinity, Mean Residence
Time for Insulin Derivatives According to the Present Invention
Data in table 1 is presented for insulin derivatives according to
the present invention (di- and trisubstituted insulins) and one
monosubstituted insulin. The affinity of the acylated insulin
analogues of this invention for the human insulin receptor was
determined by a SPA assay (Scintillation Proximity Assay)
microtiterplate antibody capture assay. Anaesthetized rats are
dosed intravenously (i.v.) with insulin analogues at various doses
and plasma concentrations of the employed compounds are measured
using immunoassays or mass spectrometry at specified intervals for
4 hours or more post-dose. Pharmacokinetic parameters where
subsequently calculated using WinNonLin Professional (Pharsight
Inc., Mountain View, Calif., USA). A. Human insulin receptor
affinity, dissociation constants (Kd) for insulin derivative
examples binding to human insulin receptor isoform A (hIRA)
relative to the value for human insulin. B. Prolongation in vivo
measured as mean residence time (MRT) upon intraveneous
administration of insulin derivative examples to rats. C.
B29N.sup..epsilon.hexadecandioyl.gamma.-L-glutamyl desB30 human
insulin is designated "C" in this list and represents a
monosubstituted reference for comparison of values only.
TABLE-US-00002 TABLE 1 A: Affinity Example for hIRA, Kd B: MRT i.v.
number (relative %) rats (h) Monosubstituted insulin* C 18.3 1.8
Insulin derivative according to the present invention 1 16.1 N/A 2
6.5 6.4 3 36.9 N/A 4 7.9 9.0 5 3.1 10.0 6 1.4 10.3 7 N/A 12.0 8 0.4
5.0 9 0.1 19.5 10 12.3 20.0 11 7.5 17.0 12 16.1 13.0 13 N/A 3.4 14
N/A N/A 15 2.6 22.0 16 0.5 N/A 17 N/A N/A 18 N/A N/A 19 N/A N/A 20
3.1 11.5 21 0.2 13.0 22 0.04 20.0 23 1.2 21.0 24 0.3 11.0 25 0.8
27.0 26 2.1 21.0 27 2.1 31.0 28 5.6 5.3 26 2.1 21.0 27 2.1 31.0 28
5.6 5.3 30 0.04 26 31 0.15 N/A 32 3.26 N/A 33 0.57 N/A 34 14.5 17
35 11.4 N/A
While certain features of the invention have been illustrated and
described herein, many modifications, substitutions, changes, and
equivalents will now occur to those of ordinary skill in the art.
It is, therefore, to be understood that the appended aspects are
intended to cover all such modifications and changes as fall within
the true spirit of the invention.
Example 30
A1N.sup..alpha.-octadecandioyl-gamma-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-gamma-L-glutamyl-OEG-OEG A14E
B16H B25H desB30 human insulin
##STR00053##
This compound was prepared in analogy with the compound of example
7 by using octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl
and A14E B16H B25H desB30 human insulin.
Product LCMS: 1767.9 Da [M+4H].sup.4+.
Calculated for C.sub.313H.sub.492N.sub.74O.sub.99S.sub.6
[M+4H].sup.4+: 1768.1 Da.
Example 31
A1N.sup..alpha.-octadecandioyl-gamma-L-glutamyl-OEG-OEG
B29N.sup..epsilon.-octadecandioyl-gamma-L-glutamyl-OEG-OEG A14E
B16H desB27 desB30 human insulin
##STR00054##
This compound was prepared in analogy with the compound of example
7 by using octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl
and A14E B16HdesB27 desB30 human insulin.
Product LCMS: 1744.8 Da [M+4H].sup.4+.
Calculated for C.sub.312H.sub.487N.sub.71O.sub.97S.sub.6
[M+4H].sup.4+: 1745.3 Da.
Example 32
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-be-
nzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminom-
ethyl-benzyl A14E B25H desB30 human insulin
##STR00055##
To a suspension of A14E B25H desB30 human insulin (50 mg) in DMSO
(1.5 ml) was added a solution of
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyde
(11.3 mg) in DMSO (0.5 mL). After 20 min, the mixture was clear and
a 1 M 2-picoline borane complex in DMSO (0.041 ml) was added. After
2.5 h and after 21 h additional 1 M 2-picoline borane complex in
DMSO (0.041 ml) was added. The mixture was purified after 24.5 h by
RP-HPLC on a C18 column using A: 0.1% trifluoroacetic acid in
water, buffer B: 0.1% trifluoroacetic acid in acetonitrile.
Gradient 20% B to 60% B over 60 minutes. Product pools were
partially evaporated in vacuo and freeze-dried providing
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-amino-
methyl-benzyl A14E B25H desB30 human insulin
LCMS: 1833.81 Da [M+4H].sup.4+. Calculated for
C.sub.332H.sub.512N.sub.74O.sub.100S.sub.6 [M+4H].sup.4+: 1834.15
Da.
MALDI: (matrix, HCCA); m/z: 7332.76 Da, calculated: 7332.60 Da
Example 33
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-be-
nzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminom-
ethyl-benzyl A14E B25H desB30 human insulin
##STR00056##
A1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-b-
enzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-amino-
methyl-benzyl A14E B25H desB30 human insulin was isolated from the
reaction mixture described in example 32.
LCMS: 1834.14 Da [M+4H].sup.4+. Calculated for
C.sub.332H.sub.512N.sub.74O.sub.100S.sub.6 [M+4H].sup.4+: 1834.15
Da.
MALDI: (matrix, HCCA); m/z: 7332.94 Da, calculated: 7332.60 Da
Example 34
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-be-
nzyl B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG
A14E desB27 desB30 human insulin
##STR00057##
This compound was prepared in analogy with the compound of example
27 by using
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-4-aminomethyl-benzaldehyd-
e and octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-succinimidyl and
A14E desB27 desB30 human insulin.
Product LCMS: 1781.8 Da [M+4H].sup.4+.
Calculated for C.sub.323H.sub.498N.sub.70O.sub.98S.sub.6
[M+4H].sup.4+: 1781.6 Da.
Example 35
B1N.sup..alpha.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-OEG-OEG-4-aminom-
ethyl-benzyl
B29N.sup..epsilon.-octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-OEG-OEG
A14E desB27 desB30 human insulin
##STR00058##
This compound was prepared in analogy with the compound of example
27 by using
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-OEG-OEG-4-aminomethyl-ben-
zaldehyde and
octadecandioyl-.gamma.-L-glutamyl-OEG-OEG-OEG-OEG-succinimidyl and
A14E desB27 desB30 human insulin.
Product LCMS: 1926.8Da [M+4H].sup.4+.
Calculated for C.sub.347H.sub.542N.sub.74O.sub.110S.sub.6
[M+4H].sup.4+: 1926.8 Da.
* * * * *